Fusogenic liposomes

ABSTRACT

The present invention relates to liposomes and virosomes and, more particularly, to liposomal and virosomal delivery systems for transporting materials such as drugs, nucleic acids and proteins.

BACKGROUND OF THE INVENTION

[0001] It is well recognized in the medical field that the mosteffective procedure for treating localized disease is to direct thepharmaceutical or drug agent (hereinafter “drugs”) to the affected area,thereby avoiding undesirable toxic effects of systemic treatment.Techniques currently being used to deliver drugs to specific targetsites within the body involve the utilization of time-release capsulesor gel matrices from which drugs slowly “leak,” or the use ofimplantable “syringes” that mechanically release drugs into muscles orinto the blood stream. Another, and perhaps more effective deliverysystem, encompasses the use of liposomes containing the appropriate drugor chemical. The liposome with encapsulated drug is directed to thespecific area of interest and, thereafter, the drug is released. Thecarrying out of this latter step is the most problematic and, in fact,the greatest barrier to the use of liposomes as drug carriers is makingthe liposomes release the drugs on demand at the target site ofinterest.

[0002] Liposomes are vesicles comprised of one or more concentricallyordered lipid bilayers which encapsulate an aqueous phase. They arenormally not leaky, but can become leaky if a hole or pore occurs in themembrane, if the membrane is dissolved or degrades, or if the membranetemperature is increased to the phase transition temperature, T_(c).Current methods of drug delivery via liposomes require that the liposomecarrier will ultimately become permeable and release the encapsulateddrug at the target site. This can be accomplished, for example, in apassive manner wherein the liposome bilayer degrades over time throughthe action of various agents in the body. Every liposome compositionwill have a characteristic half-life in the circulation or at othersites in the body and, thus, by controlling the half-life of theliposome composition, the rate at which the bilayer degrades can besomewhat regulated.

[0003] In contrast to passive drug release, active drug release involvesusing an agent to induce a permeability change in the liposome vesicle.Liposome membranes can be constructed so that they become destabilizedwhen the environment becomes acidic near the liposome membrane (see,e.g., Proc. Natl. Acad. Sci. USA 84:7851 (1987); Biochemistry 28:908(1989)). When liposomes are endocytosed by a target cell, for example,they can be routed to acidic endosomes which will destabilize theliposome and result in drug release. Alternatively, the liposomemembrane can be chemically modified such that an enzyme is placed as acoating on the membrane which slowly destabilizes the liposome. Sincecontrol of drug release depends on the concentration of enzyme initiallyplaced in the membrane, there is no real effective way to modulate oralter drug release to achieve “on demand” drug delivery. The sameproblem exists for pH-sensitive liposomes in that as soon as theliposome vesicle comes into contact with a target cell, it will beengulfed and a drop in pH will lead to drug release.

[0004] In addition to the foregoing methods, a liposome having apredetermined phase transition temperature, T_(c), above bodytemperature can be used to achieve active drug delivery. In this method,the body temperature will maintain the liposome below the T_(c) so thatthe liposome will not become leaky when placed in the body. This methodof drug release is capable of “on demand” drug delivery since suchliposomes experience a greatly increased membrane permeability at theirT_(c) which, in turn, enables drug or chemical release. To release drugsfrom such phase transition liposomes when in the body, heat must beapplied until the T_(c) is achieved. Unfortunately, the application ofheat can, in itself, create problems within the body and, frequently,the adverse effects of the heat treatment outweigh the beneficialeffects of using the liposome as a drug delivery vehicle. Moreover, suchliposomes must be made of highly purified and expensive phase transitiontemperature phospholipid materials.

[0005] In view of the foregoing, there exists a need in the art for amethod for targeted drug delivery that overcomes the disadvantages ofthe currently available methods. Specifically, a parenteral deliverysystem is required that would be stable in the circulation, followingintravenous administration, allowing retention of encapsulated orassociated drug or therapeutic agent(s). This delivery system would becapable of accumulating at a target organ, tissue or cell via eitheractive targeting (e.g., by incorporating an antibody or hormone on thesurface of the liposomal vehicle) or via passive targeting, as seen forlong-circulating liposomes. Following accumulation at the target site,the liposomal carrier would become fusogenic, without the need for anyexternal stimulus, and would subsequently release any encapsulated orassociated drug or therapeutic agent in the vicinity of the target cell,or fuse with the target cell plasma membrane introducing the drug ortherapeutic agent into the cell cytoplasm. In certain instances, fusionof the liposomal carrier with the plasma membrane would be preferredbecause this would provide more specific drug delivery and, hence,minimize any adverse effects on normal, healthy cells or tissues. Inaddition, in the case of therapeutic agents such as DNA, RNA, proteins,peptides, etc., which are generally not permeable to the cell membrane,such a fusogenic carrier would provide a mechanism whereby thetherapeutic agent could be delivered to its required intracellular siteof action. Further, by avoiding the endocytic pathway, the therapeuticagent would not be exposed to acidic conditions and/or degradativeenzymes that could inactivate said therapeutic agent. Quitesurprisingly, the present invention addresses this need by providingsuch a method.

SUMMARY OF THE INVENTION

[0006] In one embodiment, the present invention provides a fusogenicliposome comprising a lipid capable of adopting a non-lamellar phase,yet capable of assuming a bilayer structure in the presence of a bilayerstabilizing component; and a bilayer stabilizing component reversiblyassociated with the lipid to stabilize the lipid in a bilayer structure.Such fusogenic liposomes are extremely advantageous because the rate atwhich they become fusogenic can be not only predetermined, but varied asrequired over a time scale ranging from minutes to days. Control ofliposome fusion can be achieved by modulating the chemical stabilityand/or exchangeability of the bilayer stabilizing component(s).

[0007] By controlling the composition and concentration of the bilayerstabilizing component, one can control the chemical stability of thebilayer stabilizing component and/or the rate at which the bilayerstabilizing component exchanges out of the liposome and, in turn, therate at which the liposome becomes fusogenic. In addition, othervariables including, for example, pH, temperature, ionic strength, etc.can be used to vary and/or control the rate at which the liposomebecomes fusogenic.

[0008] In another embodiment, the present invention provides a methodfor delivering a therapeutic compound to a target cell at apredetermined rate, the method comprising: administering to a hostcontaining the target cell a fusogenic liposome which comprises abilayer stabilizing component, a lipid capable of adopting anon-lamellar phase, yet capable of assuming a bilayer structure in thepresence of the bilayer stabilizing component, and a therapeuticcompound or a pharmaceutically acceptable salt thereof. Administrationmay be by a variety of routes, but the therapeutic compounds arepreferably given intravenously or parenterally. The fusogenic liposomesadministered to the host may be unilamellar, having a mean diameter of0.05 to 0.45 microns, more preferably from 0.05 to 0.2 microns.

[0009] In yet another embodiment, the present invention provides alipopeptide, the lipopetide comprising (or consisting essentially of) alipid covalently attached to a peptide by means of an amide bond.Typically, the amide bond is formed between a carboxyl group of thelipid and an amino group of the peptide. In addition, the presentinvention provides a pharmaceutical composition for introducing atherapeutic compound into a cell of a host, the pharmaceuticalcomposition comprising: a liposome containing a lipopeptide, thelipopeptide comprising a lipid covalently attached to a peptide by meansof an amide bond; a therapeutic compound contained in the liposome; anda pharmaceutically acceptable carrier. Such liposomes are stable atphysiological pH, but after being internalized by cells through anendocytic pathway, the liposomes exposed to the acidic pH of theendosome are destabilized and fuse with the endosome membrane, resultingin release of their contents into the cytoplasm.

[0010] In another embodiment, the present invention provides fusogenicpH-sensitive oligomers, the oligomers having the general structures

[X—Y]_(n)

[0011] and

[X—Y—Z]_(n)

[0012] in which: X is a trifunctional substrate, wherein at least one ofthe functional groups is a carboxyl group or a protected carboxyl group;Z is a trifunctional substrate, wherein at least one of the functionalgroups is a carboxyl group or a protected carboxyl group; Y is ethyleneglycol; and n is an integer having a value ranging from 1 to 20. Inaddition, the present invention provides a pharmaceutical compositionfor introducing a therapeutic compound into a cell of a host, thepharmaceutical composition comprising: a liposome containing apH-sensitive fusogenic polymer, the pH-sensitive fusogenic polymer asdescribed above; a therapeutic compound contained in the liposome; and apharmaceutically acceptable carrier.

[0013] The present invention further provides pharmaceuticalcompositions for treatment of hosts. The compositions generally comprisea virosome having a membrane and an aqueous interior, wherein a viralmembrane fusion protein, e.g., influenza hemagglutinin protein, iscontained in the membrane, and further comprising a therapeutic compoundcontained in the virosome and a pharmaceutically acceptable carrier. Thetherapeutic compound may be carried in the aqueous interior or in themembrane of the virosome. Nucleic acids, proteins, peptides, and othercompounds may be carried in the compositions of the present invention.Generally, the hemagglutinin is derived from influenza A.

[0014] Also provided are methods for introducing therapeutic compoundsinto cells of a host. The methods typically include contacting the cellwith a virosome containing the therapeutic compound. A wide variety ofcompounds may be introduced into host cells by the present methods. Thevirosomes may be administered to the host by a variety of routes,including by parenteral, topical or inhalation administration.

[0015] Other features, objects and advantages of the invention and itspreferred embodiments will become apparent from the detailed descriptionwhich follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 illustrates the concentration dependence of bilayerstabilization by a bilayer stabilizing component (BSC). Multilamellarvesicles were prepared, as described in the examples, from mixtures ofDOPE:cholesterol:DOPE-PEG₂₀₀₀, 1:1:N, where N is the proportion ofDOPE-PEG₂₀₀₀ as indicated in the FIG. 1. ³¹P-NMR spectra were determinedat 20° C. after the sample had been allowed to equilibrate for 30minutes.

[0017]FIG. 2 illustrates the temperature dependence of bilayerstabilization by BSC. Multilamellar vesicles were prepared, as describedin the examples, from mixtures of DOPE:cholesterol:DOPE-PEG₂₀₀₀ at aratio of: A, 1:1:0.1; or B, 1:1:0.25. The samples were cooled to 0° C.and ³¹P-NMR spectra were determined from 0° C. to 60° C. at 10° C.intervals. The samples were allowed to equilibrate at each temperaturefor 30 min. prior to data accumulation.

[0018]FIG. 3 illustrates the effect of headgroup size on the bilayerstabilizing ability of BSC. Multilamellar vesicles were prepared fromeither A, DOPE:cholesterol:DOPE-PEG₂₀₀₀, 1:1:0.05, or B,DOPE:cholesterol:DOPE-PEG₅₀₀₀, 1:1:0.05. Other conditions were thesame-as for FIG. 2.

[0019]FIG. 4 illustrates the effect of the acyl chain composition on thebilayer stabilizing ability of BSC. Multilamellar vesicles wereprepared, as described in the examples, from either A,DOPE:cholesterol:DMPE-PEG₂₀₀₀, 1:1:0.1, B,DOPE:cholesterol:DPPE-PEG₂₀₀₀, 1:1:0.1, or C,DOPE:cholesterol:DSPE-PEG₂₀₀₀, 1:1:0.1. Other conditions were the sameas for FIG. 2.

[0020]FIG. 5 illustrates the ability of PEG-Ceramide to act as a bilayerstabilizing component. Multilamellar vesicles were prepared, asdescribed in the examples, from DOPE:cholesterol:egg ceramide-PEG₂₀₀₀ ata ratio of A, 1:1:0.1 or B, 1:1:0.25. Other conditions were the same asfor FIG. 2.

[0021]FIG. 6 illustrates the freeze-fracture electron micrograph of MLVsprepared from DOPE:cholesterol:DOPE-PEG₂₀₀₀ (1:1:0.1). The samples wereprepared as described in the examples. The bar represents 500 nm.

[0022]FIG. 7 illustrates the freeze-fracture electron micrograph of LUVsprepared from DOPE:cholesterol:DOPE-PEG₂₀₀₀ (1:1:0.1). The samples wereprepared as described in the examples. The bar represents 500 nm.

[0023]FIG. 8 illustrates the elution profiles of LUVs prepared fromDOPE:cholesterol:DSPE-PEG₂₀₀₀, and micelles composed of DSPE-PEG₂₀₀₀.LUVs were prepared, as described in the examples, fromDOPE:cholesterol:DSPE-PEG₂₀₀₀ (1:1:0.1) with trace amounts of ¹⁴C-DPPC(Δ) and ³H-DSPE-PEG₂₀₀₀.() They were chromatographed as described inthe examples. In a separate experiment, micelles were prepared fromDSPE-PEG₂₀₀₀ labelled with ³H-DSPE-PEG₂₀₀₀ (◯) and chromatographed onthe same Sepharose 4B column.

[0024]FIG. 9 illustrates the inhibition of fusion by PEG-PE. Liposomeswere prepared from equimolar mixtures of DOPE and POPS containing (a) 0;(b) 0.5; (c) 1 or (d) 2 mol % DMPE-PEG₂₀₀₀. In addition to the abovelipids, labelled liposomes also contained the fluorescent lipids NBD-PEand Rh-PE at 0.5 mol %. Fluorescently labelled liposomes (finalconcentration 60 μM) were incubated at 37° C. for 30 sec. before theaddition of a three-fold excess of unlabelled liposomes, followed oneminute later by CaCl₂ (final concentration 5 mM).

[0025]FIG. 10 illustrates the recovery of fusogenic activity afterPEG-PE removal. Fusion between fluorescently labelled and unlabelledliposomes containing 2 mol % DMPE-PEG₂₀₀₀ was assayed as described underFIG. 9, except that one minute after addition of CaCl₂, a 12-fold excess(over labelled vesicles) of POPC liposomes (curve a) or an equivalentvolume of buffer (curve b) was added.

[0026]FIG. 11 illustrates the concentration dependence of recovery offusogenic activity. Fusion between fluorescently labelled and unlabelledliposomes containing (a) 2; (b) 3; (c) 5 or (d) 10 mol % DMPE-PEG₂₀₀₀was assayed as described under FIG. 10, except that POPC liposomes wereadded as a 36-fold excess over labelled vesicles.

[0027]FIG. 12 illustrates programmable fusion. Fusion betweenfluorescently labelled and unlabelled liposomes containing 2 mol % ofthe indicated PE-PEG₂₀₀₀ was assayed as described under FIG. 10. The %fusion was calculated as described in the examples. FIG. 12A:DMPP-PEG₂₀₀₀ (); DPPE-PEG₂₀₀₀ (♦); DSPE-PEG₂₀₀₀ (▴); and FIG. 12B:DOPE-PEG₂₀₀₀ (▴), egg ceramide-PEG₂₀₀₀ (▾).

[0028]FIG. 13 illustrates the effect of PEG molecular weight on fusion.FIG. 13A: Assays were carried out as described in FIG. 9 using liposomeswhich contained (a) 0; (b) 0.25; (c) 0.5 or (d) 1 mol % DMPE-PEG₅₀₀₀;and FIG. 13B: Assays were performed as described under FIG. 12 usingliposomes which contained 1 mol % DMPE-PEG₅₀₀₀ (); DPPE-PEG₅₀₀₀ (♦) orDSPE-PEG₅₀₀₀ (▴).

[0029]FIG. 14 illustrates the comparison of PEG₂₀₀₀ to PEG₅₀₀₀ at equalconcentration of oxyethylene groups. Liposomes contained either 2 mol %PEG₅₀₀₀ (upper curve) or 5 mol % PEG₂₀₀₀ (lower curve). Other conditionswere as described under FIG. 11.

[0030]FIG. 15 illustrates the effect of salt concentration on fusion ofDOPE:DODAC Liposomes. Liposomes were prepared from DOPE:DODAC (85:15).Donor liposomes also contained the fluorescent lipids, NBD-PE and Rh-PEat 0.5 mol %. Donor liposomes (final concentration 60 μM) were incubatedat 37° C. for 30 sec. before the addition of a three-fold excess ofunlabelled acceptor liposomes followed 1 min later by NaCl to give theindicated final concentration.

[0031]FIG. 16 illustrates the inhibition of fusion of DOPE:DODACliposomes by PEG-PE. Liposomes were prepared from either DOPE:DODAC(85:15) or DOPE:DODAC:DMPE-PEG₂₀₀₀ (83:15:2). Fusion was assayed asdescribed under FIG. 1 using 300 mM NaCl.

[0032]FIG. 17 illustrates the recovery fusogenic activity after PEGremoval. Liposomes were prepared from eitherDOPE:DODAC:ceramide(C8:0)-PEG₂₀₀₀, 83:15:2 orDOPE:cholesterol:ceramide(C8:0)-PEG₂₀₀₀, 38:45:15:2. Fusion was assayedas described under FIG. 2 except that at the indicated times a 30 foldexcess (over donors) of liposomes composed of POPC or POPC:cholesterol(55:45) was added.

[0033]FIG. 18 illustrates the effect of the lipid anchor on the rate ofPEG-lipid removal. Fluorescently labelled and unlabelled liposomes wereprepared from DOPE:DODAC:PEG-lipid, 83:15:2, using DMPE-PEG₂₀₀₀ (),ceramide(egg)-PEG₂₀₀₀ or (C14:0) ceramide-PEG₂₀₀₀ (♦). Labelledliposomes were mixed with a 3 fold excess of unlabelled liposomes and300 mM NaCl in a cuvette in a dark water bath at 37° C. At zero time a13-fold excess (over labelled vesicles) of POPC liposomes was added andthe fluorescence intensity was measured at the indicated times. At theend of the assay Triton X-100 (0.5% final) was added to eliminate energytransfer and the % fusion was calculated from the change in efficiencyof energy transfer. Maximum fusion was calculated from a standard curveof energy transfer efficiency against the molar fraction of Rh-PE in themembrane assuming complete mixing of labelled and unlabelled liposomes.

[0034]FIG. 19 illustrates the inhibition of fusion betweenDOPE:cholesterol:DODAC liposomes and anionic liposomes by PEG-ceramide.Liposomes were prepared from DOPE:cholesterol:DODAC, 40:45:15 (no PEG)or DOPE:cholesterol:DODAC:(C14:0) ceramide-PEG₂₀₀₀, 36:45:15:4 (4% PEG).Acceptor liposomes were prepared from DOPE:cholesterol:POPS, 25:45:30. Athree-fold excess of acceptors was added to labelled vesicles after 30sec. and the fluorescence monitored at 517 nm with excitation at 465 nm.

[0035]FIG. 20 illustrates the recovery of fusogenic activity upon PEGremoval. Donor liposomes (50 μM) were prepared fromDOPE:cholesterol:DODAC:(C14:0)ceramide-PEG₂₀₀₀, 36:45:15:4 and mixedwith acceptor liposomes (150 μM) prepared from DOPE:cholesterol:POPS,25:45:30. At zero time either 1 mM POPC:cholesterol liposomes (▴) or anequivalent volume of buffer () was added. Fluorescence was monitored at517 nm with excitation at 465 nm.

[0036]FIG. 21 illustrates the inhibition of fusion betweenDOPE:cholesterol:DODAC liposomes and erythrocyte ghosts by,PEG-ceramide. Liposomes were prepared from DOPE:cholesterol:DODAC,40:45:15 (no PEG) or DOPE:cholesterol:DODAC:(C14:0)ceramide-PEG₂₀₀₀,36:45:15:4 (4% PEG). Ghosts (50 μM phospholipid) were added to donors(50 μM total lipid) after 30 sec. and the fluorescence monitored at 517nm with excitation at 465 nm.

[0037]FIG. 22 illustrates the fusion of fluorescent liposomes composedof DOPE:cholesterol:DODAC (40:45:15) orDOPE:cholesterol:DODAC:PEG-ceramide (35:45:15:5). LUVs composed ofDOPE:cholesterol:DODAC (40:45:15) fused with RBCs (panels a and b);incorporation of PEG-ceramide (C8:0) into the LUVs at 5 mol % blockedfusion (panels c and d); however, when an exogenous sink for thePEG-ceramide was included, fusogenic activity was recovered withinminutes (panels e and f). Panels a, c and e are views under phasecontrast, and panels b,d and f are the same fields view underfluorescent light.

[0038]FIG. 23 illustrates the results when PEG-ceramides with longerfatty amide chains (C14:0) are used and the liposomes are pre-incubationwith an exogenous sink prior to the addition of the RBCs. No fusion wasobserved after pre-incubation of the fusogenic LUVs with the sink forfive minutes prior to addition of RBC (panels a and b); after a 1 hourpre-incubation, some fusion with RBCs was observed (panels c and d);however, with longer incubations times (2 hours), the pattern offluorescent labeling changed and extensive punctate fluorescence wasobserved (panels e and f). Panels a, c and e are views under phasecontrast, and panels b,d and f are the same fields view underfluorescent light.

[0039]FIG. 24 illustrates the results when PEG-ceramides with longerfatty amide chains (C20:0) are used and the liposomes are preincubationwith an exogenous sink prior to the addition of the RBCs. No fusion wasobserved after pre-incubation of the LUVs with the sink for five minutes(panels a and b), 1 hour (panels c and d) or 2 hours (panels e and f).Panels a, c and e are views under phase contrast, and panels b,d and fare the same fields view under fluorescent light.

[0040]FIG. 25 graphically illustrates the fusion of PEG₂₀₀₀-DMPE andPEG₂₀₀₀-Ceramide (C14:0) containing vesicles with an anionic target.

[0041]FIG. 26 graphically shows the effect of increasing concentrationsof PEG-Ceramide (C20) on liposome clearance from the blood. ³H-labeledliposomes composed of DOPE (dioleoylphosphatidylethanolamine), 15 mol %DODAC (N,N-dioleoyl-N,N-dimethylammonium chloride) and the indicatedconcentrations of PEG-Ceramide (C20) were injected i.v. into mice.Biodistribution was examined at 1 hour after injection, and the datawere expressed as a percentage of the injected dose in the blood (upperpanel) and liver (lower panel) with SD (standard deviation) (n=3).

[0042]FIG. 27 graphically illustrates the effect of increasingconcentrations of DODAC on the biodistribution of liposomes in theblood. ³H-labeled liposomes composed of DOPE, 10 (open squares) or 30(open triangles) mol % PEG-Ceramide (C20), and the indicatedconcentration of DODAC were injected i.v. into mice. Biodistribution wasexamined at 1 hour after injection, and the data were expressed as apercentage of the injected dose in the blood.

[0043]FIG. 28 graphically shows the liposome levels in the blood andliver at different times after injection. ³H-labeled liposomes composedof DOPE/DODAC (85:15 mol/mol) (open circles with 0% PEG-Ceramide (C20)),DOPE/DODAC/PEG-Ceramide (C20) (75:15:10 mol/mol/mol) (open squares with10% PEG-Ceramide (C20)), and DOPE/DODAC/PEG-Ceramide (C20) (55:15:30mol/mol/mol) (open triangles with 30% PEG-Ceramide (C20)) were injectedi.v. into mice. Biodistribution was examined at indicated times, and thedata were expressed as a percentage of the injected dose in the blood(FIG. 28A) and in the liver (FIG. 28B) with SD (n=3).

[0044]FIG. 29 illustrates that the fusion of reconstituted influenzavirosomes with erythrocyte membranes is dependent on low pH.

[0045]FIG. 30 illustrates the fusion of influenza virosomes from withinBHK cell endosomes as monitored by a decrease of pyrene excimerfluorescence and the blocking of fusion by NH₄Cl, an inhibitor ofendosomal acidification.

[0046]FIG. 31 illustrates that delivery of diphtheria toxin A chainencapsulated in fusogenic virosomes induces complete inhibition of thecellular protein synthesis in BHK-21 cells, whereas free DTA or emptyvirosomes have no effect on protein synthesis, and that the effect ofvirosome-encapsulated DTA is blocked completely by NH₄Cl, or bypretreatment of the virosomes at low pH causing an irreversibleinactivation of their fusion activity.

[0047]FIG. 32 illustrates the time course of gelonin delivery to BHKcells, as mediated by influenza virosomes fusing from within endosomes.

[0048]FIG. 33 depicts gelonin delivery to BHK-21 cells mediated byinfluenza virosomes fusing from within endosomes.

[0049]FIG. 34 demonstrates that influenza virosomes can fuse with theplasma membrane of BHK cells, thereby mediating intracellular deliveryof encapsulated gelonin.

[0050]FIG. 35 illustrates titration of gelonin-mediated inhibition ofprotein synthesis to a level corresponding to a single virosome fusingper cell.

[0051]FIG. 36 shows the expression of β-Gal in transfected BHK cells asa function of % DODAC in the fusion protein TCS.

[0052]FIG. 37 shows the DNA binding capacity of the virosomes containing30 mol % DODAC, where increasing amounts of ³H-pCMVβ-gal were added tovirosomes and, after incubation and centrifugation, radioactivitydetermined in the pellet (, ▪) and in the supernatant (▴, ▾) of twoindependent experiments.

[0053]FIG. 38 shows the expression of β-Gal in transfected BHK cells asa function of DNA added per well complexed to virosomes.

[0054]FIG. 39 illustrates the amino acid sequence of the hemagglutininHA2 subunit N-terminal “fusion peptide” of influenza virus X31 strain(wt), sequence of the E4 peptide prepared by Rafalski, et al.(Biochemistry, 30:10211-10220 (1991)) containing a glutamic acidsubstitution at position 4, and sequence of the peptide AcE4K used inthis study, including N-terminal acetylation and the addition oflysine-21 at the C-terminus. A representation of AcE4K as an α-helixwith acidic sidechains shown in black and hydrophobic residues in whitedemonstrates the potential amphipathic nature of the peptide in thisconformation.

[0055]FIG. 40 sets forth the structure and synthesis of the Lipo-AcE4Klipopeptide (4). The activated lipid (3) reacts exclusively with theprimary amine on the C-terminal lysine of the peptide.

[0056]FIG. 41 illustrates the effects of pH and the presence of lipidvesicles on the secondary structure of AcE4K and Lipo-AcE4K. FIG. 41A:CD spectra of 25 μM AcE4K in 10 mM phosphate buffer at pH 7.5 (A, C) andpH 5.0 (B, D) in the absence (A, B) or presence (C, D) of POPC LUVs, 2.5mM lipid. FIG. 41B: CD spectra of POPC LUVs (2.5 mM lipid) prepared with1 mol % Lipo-AcE4K in phosphate buffer at pH 7.5 or pH 5.0. All spectrarepresent the average of 5 scans from which buffer and lipid signal hasbeen subtracted, as appropriate.

[0057]FIG. 42 sets forth the tryptophan fluorescence emission spectra ofAcE4K and Lipo-AcE4K showing the effects of pH and the presence of lipidvesicles: FIG. 42A: 1 μM AcE4K in 10 mM phosphate buffer at pH 7.5 (A,C) and pH 5.0 (B, D) in the absence (A, B) or presence (C, D) of POPCLUVs, 0.1 mM lipid; FIG. 42B: POPC LUVs (0.1 mM lipid) prepared with 1mol % Lipo-AcE4K in phosphate buffer at pH 7.5 or pH 5.0. Spectra werecorrected by subtracting scans of phosphate buffer or LUVs, asappropriate.

[0058]FIG. 43 illustrates the effect of pH on lipid mixing for 5 mol %Lipo-AcE4K in POPC LUVs. Lipopeptide was added from a 2 mM DMSO stocksolution to a 1:3 mixture of labelled and unlabelled vesicles prior tothe addition of 1 M HCl to achieve the indicated pH.

[0059]FIG. 44 illustrates the effects of Lipo-AcE4K concentration onlipid mixing and leakage in POPC LUVs. FIG. 44A: Varying amounts oflipopeptide were added to a 1:3 mixture of labeled and unlabeledvesicles (0.2 mM total lipid) from a 2 mM stock solution in DMSO, suchthat the final DMSO concentration was less than 1% by volume. Lipidmixing assays were as described above, adding 1 M HCl at 30 seconds toachieve a final pH of 5.0. FIG. 44B: Vesicles containing 6 mM ANTS and75 mM DPX dissolved in HMA buffer, pH 7.5, were diluted to 0.2 mM lipidprior to addition of varying amounts of lipopeptide from a 2 mM DMSOstock solution. At 30 seconds, 1 M HCl was added to achieve a pH of 5.0.

[0060]FIG. 45 illustrates the exchange of Lipo-AcE4K between vesiclepopulations and lipid mixing with membranes lacking lipopeptide. FIG.45A: 10 mol % Lipo-AcE4K was added to prepared POPC LUVs, and the amountof peptide associated with POPC vesicles was determined by micro-BCAassay before and after incubation with POPC MLVs. A control experimentusing LUVs without lipopeptide is also known. Assays were carried out induplicate, and deviations from means were negligible except where errorbars are shown. FIG. 45B: Lipid mixing assay were performed afterpreincubation of selected liposome populations with sufficientLipo-AcE4K to achieve a 5 mol % concentration in liposomal outermonolayers. The lipopeptide was included in both fluorescently labeledand unlabeled populations (A), in labeled vesicles only (B), inunlabeled vesicles only (C), or in neither labeled nor labeled liposomes(D).

[0061]FIG. 46 illustrates the lipid mixing and leakage in EPC:Chol(55:45) LUVs. FIG. 46A: 0 to 10 mol % Lipo-AcE4K was added to a 1:3mixture of labeled and unlabeled vesicles (0.2 mM total lipid) from a 2mM stock solution in DMSO. Lipid mixing assays were as described above,adding 1 M HCl at 30 seconds to achieve a final pH of 5.0. FIG. 46B:Leakage assays from 0 to 10 mol % Lipo-AcE4K added to EPC/Chol (55:45)liposomes containing 6 mM ANTS and 75 mM DPX. For comparison,corresponding assays for 10 mol % of the free peptide AcE4K is alsoshown (dotted lines).

[0062]FIG. 47 illustrates the effect of transbilayer distribution ofLipo-AcE4K on lipid mixing in EPC/Chol (55:45) LUVs. 10 mol % Lipo-AcE4Lwas either present in the outer monolayer of liposomes (B, C) or in bothmonolayers (D, E) and was present in only the fluorescently labelled LUVpopoulation (B, D) or in both labelled and unlabelled LUVs (C, E). Acontrol lipid mixing assay where neither vesicle population containedlipopeptide is also shown (A).

[0063]FIG. 48 sets forth the freeze-fracture electron micrographs ofEPC/Chol liposomes: effect of Lipo-AcE4K and pH. EPC/Chol (55:45) LUVswere prepared with (C, D-F) and without (A, B) 10 mol % Lipo-AcE4K at atotal lipid concentration of 5 mM in HMA buffer. Platinum-carbonreplicas were prepared at pH 7.5 (A, C) or 5 minutes followingacidification to pH 5.0 by the addition of 1 M HCl (B, D-F). Originalmagnification was 20,000×, and bars represent 200 nm.

[0064]FIG. 49 illustrates the effect of method of lipopeptideincorporation into EPC/Chol (55:45) LUVs on lipid mixing witherythrocyte membranes. FIG. 49A: 10 mol % Lipo-AcE4K was added from DMSOstock solution to fluorescently labeled liposomes and pre-incubated at25° C. for 5 minutes prior to addition of erythrocyte membranes. Thelipopeptide is present only in the outer monolayer of vesicles. FIG.49B: Lipid-mixing assays of co-lyophilized 10 mol % Lipo-AcE4K inEPC/Chol (55:45) with erythrocyte ghosts. The lipopeptide is present ininner and outer monolayers of the fluorescently labeled vesicles.

[0065]FIG. 50 sets forth the fluorescence micrographs showing theappearance of Rh-PE in erythrocyte membranes upon lipid mixing with 10mol % Lipo-AcE4K in EPC/Chol (55:45). Liposomes were prepared from aco-lyophilized preparation of 10 mol % Lipo-AcE4K in EPC/Chol containing0.5 mol % each of NBD-PE and Rh-PE. Liposome and erythrocyte membraneswere mixed in a 1:3 lipid ratio (1 mM total lipid): (A) phase contrastand (B) Rh-PE fluorescence at pH 7.5; (C) phase contrast and (D) Rh-PEfluorescence after reducing pH to 5.0.

[0066]FIG. 51 illustrates a reaction scheme which can be used tosynthesize the basic units for preparing glutamic acid-tetraethyleneglycol oligomers.

[0067]FIG. 52 illustrates the synthetic sequence for the chain extensionto prepare the oligomer [Glu-TEG]_(n).

[0068]FIG. 53 illustrates the synthetic sequence for the chain extensionto prepare the oligomer [Glu-TEG-Glu]_(n).

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS Contents

[0069] I. Glossary

[0070] II. Fusogenic Liposomes Containing Bilayer Stabilizing Components

[0071] III. Fusogenic Liposomes Containing Fusogenic Peptides

[0072] IV. Fusogenic Liposomes Containing Fusogenic Polymers

[0073] V. Methods of Preparing Liposomes

[0074] VI. Virosome-Mediated Intracellular Delivery of TherapeuticAgents

[0075] VII. Examples

[0076] I. Glossary

[0077] The term “acyl” refers to a radical produced from an organic acidby removal of the hydroxyl group. Examples of acyl radicals includeacetyl, pentanoyl, palmitoyl, stearoyl, myristoyl, caproyl and oleoyl.

[0078] The term “lipid” refers to any suitable material resulting in abilayer such that a hydrophobic portion of the lipid material orientstoward the bilayer while a hydrophilic portion orients toward theaqueous phase. Amphipathic lipids are necessary as the primary lipidvesicle structural element. Hydrophilic characteristics derive from thepresence of phosphato, carboxylic, sulfato, amino, sulfhydryl, nitro,and other like groups. Hydrophobicity could be conferred by theinclusion of groups that include, but are not limited to, long chainsaturated and unsaturated aliphatic hydrocarbon groups and such groupssubstituted by one or more aromatic, cycloaliphatic or heterocyclicgroup(s). The preferred amphipathic compounds are phosphoglycerides andsphingolipids, representative examples of which includephosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoylphosphatidylcholine ordilinoleoylphosphatidylcholine could be used. Other compounds lacking inphosphorus, such as sphingolipid and glycosphingolipid families are alsowithin the group designated as lipid. Additionally, the amphipathiclipids described above may be mixed with other lipids includingtriglycerides and sterols.

[0079] The term “neutral lipid” refers to any of a number of lipidspecies which exist either in an uncharged or neutral zwitterionic format physiological pH. Such lipids include, for examplediacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, and cerebrosides.

[0080] The term “non-cationic lipid” refers to any neutral lipid asdescribed above as well as anionic lipids. Examples of anionic lipidsinclude cardiolipin, diacylphosphatidylserine and diacylphosphatidicacid.

[0081] The term “cationic lipid” refers to any of a number of lipidspecies which carry a net positive charge at physiological pH. Suchlipids include, but are not limited to, DODAC, DOTMA, DDAB, DOTAP,DC-Chol and DMRIE. Additionally, a number of commercial preparations ofcationic lipids are available which can be used in the presentinvention. These include, for example, LIPOFECTIN® (commerciallyavailable cationic liposomes comprising DOTMA and DOPE, from GIBCO/BRL,Grand Island, N.Y., USA); LIPOFECTAMINE® (commercially availablecationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL); andTRANSFECTAM® (commercially available cationic lipids comprising DOGS inethanol from Promega Corp., Madison, Wis., USA).

[0082] The term “transfection” as used herein, refers to theintroduction of polyanionic materials, particularly nucleic acids, intocells. The term “lipofection” refers to the introduction of suchmaterials using liposome complexes. The polyanionic materials can be inthe form of DNA or RNA which is linked to expression vectors tofacilitate gene expression after entry into the cell. Thus, thepolyanionic material used in the present invention is meant to includeDNA having coding sequences for structural proteins, receptors andhormones, as well as transcriptional and translational regulatoryelements (i.e., promoters, enhancers, terminators and signal sequences)and vector sequences. Methods of incorporating particular nucleic acidsinto expression vectors are well known to those of skill in the art, butare described in detail in, for example, Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring HarborLaboratory, (1989) or Current Protocols in Molecular Biology, F.Ausubel, et al., ed. Greene Publishing and Wiley-Interscience, New York(1987), both of which are incorporated herein by reference.

[0083] “Peptides,” “polypeptides” and “oligopeptides” are chains ofamino acids (typically L-amino acids) whose a carbons are linked throughpeptide bonds formed by a condensation reaction between the carboxylgroup of the α carbon of one amino acid and the amino group of the αcarbon of another amino acid. The terminal amino acid at one end of thechain (i.e., the amino terminal) has a free amino group, while theterminal amino acid at the other end of the chain (i.e., the carboxyterminal) has a free carboxyl group. As such, the term “amino terminus”(abbreviated N-terminus) refers to the free α-amino group on the aminoacid at the amino terminal of the peptide, or to the α-amino group(imino group when participating in a peptide bond) of an amino acid atany other location within the peptide. Similarly, the term “carboxyterminus” (abbreviated C-terminus) refers to the free carboxyl group onthe amino acid at the carboxy terminus of a peptide, or to the carboxylgroup of an amino acid at any other location within the peptide.

[0084] Typically, the amino acids making up a polypeptide are numberedin order, starting at the amino terminal and increasing in the directionof the carboxy terminal of the polypeptide. Thus, when one amino acid issaid to “follow” another, that amino acid is positioned closer to thecarboxy terminal of the polypeptide than the “preceding” amino acid.

[0085] The term “residue” is used herein to refer to an amino acid or anamino acid mimetic that is incorporated into a polypeptide by an amidebond or an amide bond mimetic. As such, the amino acid may be anaturally occurring amino acid or, unless otherwise limited, mayencompass known analogs of natural amino acids that function in a mannersimilar to the naturally occurring amino acids (i.e., amino acidmimetics). Moreover, an amide bond mimetic includes peptide backbonemodifications well known to those skilled in the art.

[0086] The phrase “consisting essentially of” is used herein to excludeany elements that would substantially alter the essential properties ofthe fusogenic peptides to which the phrase refers. Thus, the descriptionof a polypeptide “consisting essentially of . . . ” excludes any aminoacid substitutions, additions, or deletions that would substantiallyalter the biological activity of that peptide.

[0087] The amino acids referred to herein are described by shorthanddesignations as follows: TABLE I Amino Acid Nomenclature Name 3-letter 1letter Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp DCysteine Cys C Glutamic Acid Glu E Glutamine Gln Q Glycine Gly GHistidine His H Homoserine Hse — Isoleucine Ile I Leucine Leu L LysineLys K Methionine Met M Methionine sulfoxide Met (O) — Methioninemethylsulfonium Met (S-Me) — Norleucine Nle — Phenylalanine Phe FProline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine TyrY Valine Val V

[0088] II. Fusogenic Liposomes Containing Bilayer Stabilizing Components

[0089] In one embodiment of the present invention, a fusogenic liposomeis provided, the fusogenic liposome comprising: a lipid capable ofadopting a non-lamellar phase, yet capable of assuming a bilayerstructure in the presence of a bilayer stabilizing component; and abilayer stabilizing component reversibly associated with the lipid tostabilize the lipid in a bilayer structure. Such fusogenic liposomes areadvantageous because the rate at which they become fusogenic can be notonly predetermined, but varied as required over a time scale of a fewminutes to several tens of hours. It has been found, for example, thatby controlling the composition and concentration of the, bilayerstabilizing component, one can control the rate at which the bilayerstabilizing component exchanges out of the liposome and, in turn, therate at which the liposome becomes fusogenic.

[0090] The polymorphic behavior of lipids in organized assemblies can beexplained qualitatively in terms of the dynamic molecular shape concept(see, Cullis, et al., in “Membrane Fusion” (Wilschut, J. and D. Hoekstra(eds.), Marcel Dekker, Inc., New York, (1991)). When the effectivecross-sectional areas of the polar head group and the hydrophobic regionburied within the membrane are similar then the lipids have acylindrical shape and tend to adopt a bilayer conformation. Cone-shapedlipids which have polar head groups that are small relative to thehydrophobic component, such as unsaturated phosphatidylethanolamines,prefer non-bilayer phases such as inverted micelles or inverse hexagonalphase (H_(II)). Lipids with head groups that are large relative to theirhydrophobic domain, such as lysophospholipids, have an inverted coneshape and tend to form micelles in aqueous solution. The phasepreference of a mixed lipid system depends, therefore, on thecontributions of all the components to the net dynamic molecular shape.As such, a combination of cone-shaped and inverted cone-shaped lipidscan adopt a bilayer conformation under conditions where either lipid inisolation cannot (see, Madden and Cullis, Biochim. Biophys. Acta,684:149-153 (1982)).

[0091] A more formalized model is based on the intrinsic curvaturehypothesis (see, e.g., Kirk, et al., Biochemistry, 23:1093-1102 (1984)).This model explains phospholipid polymorphism in terms of two opposingforces. The natural tendency of a lipid monolayer to curl and adopt itsintrinsic or equilibrium radius of curvature (R_(o)) which results in anelastically relaxed monolayer is opposed by the hydrocarbon packingconstraints that result. Factors that decrease the intrinsic radius ofcurvature, such as increased volume occupied by the hydrocarbon chainswhen double bonds are introduced, tend to promote H_(II) phaseformation. Conversely, an increase in the size of the headgroupincreases R_(o) and promotes bilayer formation or stabilization.Introduction of apolar lipids that can fill the voids between invertedlipid cylinders also promotes H_(II) phase formation (see, Gruner, etal., Proc. Natl. Acad. Sci. USA, 82:3665-3669 (1989); Sjoland, et al.,Biochemistry, 28:1323-1329 (1989)).

[0092] Lipids which can be used to form the fusogenic liposomes of thepresent invention are those which adopt a non-lamellar phase underphysiological conditions or under specific physiological conditions,e.g., in the presence of calcium ions, but which are capable of assuminga bilayer structure in the presence of a bilayer stabilizing component.Such lipids include, but are not limited to, phosphatidylenthanolamines,ceramides, glycolipids, or mixtures thereof. Other lipids known to thoseof skill in the art to adopt a non-lamellar phase under physiologicalconditions can also be used. Moreover, it will be readily apparent tothose of skill in the art that other lipids can be induced to adopt anon-lamellar phase by various non-physiological changes including, forexample, changes in pH or ion concentration (e.g., in the presence ofcalcium ions) and, thus, they can also be used to form the fusogenicliposomes of the present invention. In a presently preferred embodiment,the fusogenic liposome is prepared from a phosphatidylethanolamine. Thephosphatidylethanolamine can be saturated or unsaturated. In a presentlypreferred embodiment, the phosphatidylyethanolamine is unsaturated. Inan equally preferred embodiment, the fusogenic liposome is prepared froma mixture of a phosphatidylethanolamine (saturated or unsaturated) and aphosphatidylserine. In another equally preferred embodiment, thefusogenic liposome is prepared from a mixture of aphosphatidylethanolamine (saturated or unsaturated) and a cationiclipid.

[0093] Examples of suitable cationic lipids include, but are not limitedto, the following: DC-C hol, 3β-(N-(N′,N′-dimethylaminoethane)carbamoyl) cholesterol (see, Gao, et al.,Biochem. Biophys. Res. Comm. 179:280-285 (1991); DDAB,N,N-distearyl-N,N-dimethylammonium bromide; DMRIE,N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide; DODAC, N,N-dioleyl-N,N-dimethylammonium chloride (see, commonlyowned U.S. patent application Ser. No. 08/316,399, filed Sep. 30, 1994,which is incorporated herein by reference); DOGS,diheptadecylamidoglycyl spermidine; DOSPA,N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate; DOTAP,N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; andDOTMA, N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride. Ina presently preferred embodiment, N,N-dioleoyl-N,N-dimethylammoniumchloride is used in combination with a phosphatidylethanolamine.

[0094] In accordance with the present invention, lipids adopting anon-lamellar phase under physiological conditions can be stabilized in abilayer structure by bilayer stabilizing components which are eitherbilayer forming themselves, or which are of a complementary dynamicshape. The non-bilayer forming lipid is stabilized in the bilayerstructure only when it is associated with, i.e., in the presence of, thebilayer stabilizing component. In selecting an appropriate bilayerstabilizing component, it is imperative that the bilayer stabilizingcomponent be capable of transferring out of the liposome, or of beingchemically modified by endogenous systems such that, with time, it losesits ability to stabilize the lipid in a bilayer structure. Only whenliposomal stability is lost or decreased can fusion of the liposome withthe plasma membrane of the target cell occur. The bilayer stabilizingcomponent is, therefore, “reversibly associated” with the lipid and onlywhen it is associated with the lipid is the lipid constrained to adoptthe bilayer structure under conditions where it would otherwise adopt anon-lamellar phase. As such, the bilayer stabilizing components of thepresent invention must be capable of stabilizing the lipid in a bilayerstructure, yet they must be capable of exchanging out of the liposome,or of being chemically modified by endogenous systems so that, withtime, they lose their ability to stabilize the lipid in a bilayerstructure, thereby allowing the liposome to become fusogenic.

[0095] Examples of suitable bilayer stabilizing components include, butare not limited to, lipid, lipid-derivatives, detergents, proteins andpeptides. In a presently preferred embodiment, the bilayer stabilizingcomponent is polyethyleneglycol conjugated to, i.e., coupled to, aphosphatidylethanolamine. In an equally preferred embodiment, thebilayer stabilizing component is polyethyleneglycol conjugated to aceramide. Polyethyleneglycol can be conjugated to aphosphatidylethanolamine or, alternatively, to a ceramide using standardcoupling reactions known to and used by those of skill in the art. Inaddition, preformed polyethyleneglycol-phosphatidylethanolamineconjugates are commercially available from Avanti Polar Lipids(Alabaster, Ala.).

[0096] Polyethyleneglycols of varying molecular weights can be used toform the bilayer stabilizing components of the present invention.Polyethyleneglycols of varying molecular weights are commerciallyavailable from a number of different sources or, alternatively, they canbe synthesized using standard polymerization techniques well-known tothose of skill in the art. In a presently preferred embodiment, thepolyethylene glycol has a molecular weight ranging from about 200 toabout 10,000, more preferably from about 1,000 to about 8,000, and evenmore preferably from about 2,000 to about 6,000. Generally, it has-beenfound that increasing the molecular weight of the polyethyleneglycolreduces the concentration of the bilayer stabilizing component requiredto achieve stabilization.

[0097] Phosphatidylethanolamines having a variety of acyl chain groupsof varying chain lengths and degrees of saturation can be conjugated topolyethyleneglycol to form the bilayer stabilizing component. Suchphosphatidylethanolamines are commercially available, or can be isolatedor synthesized using conventional techniques known to those of skill inthe art. Phosphatidylethanolamines containing saturated or unsaturatedfatty acids with carbon chain lengths in the range of C₁₀ to C₂₀ arepreferred. Phosphatidylethanolamines with mono- or diunsaturated fattyacids and mixtures of saturated and unsaturated fatty acids can also beused. Suitable phosphatidylethanolamines include, but are not limitedto, the following: dimyristoylphosphatidylethanolamine (DMPE),dipalmitoylphosphatidylethanolamine (DPPE),dioleoylphosphatidylethanolamine (DOPE) anddistearoylphosphatidyl-ethanolamine (DSPE).

[0098] As with the phosphatidylethanolamines, ceramides having a varietyof acyl chain groups of varying chain lengths and degrees of saturationcan be coupled to polyethyleneglycol to form the bilayer stabilizingcomponent. It will be apparent to those of skill in the art that incontrast to the phosphatidylethanolamines, ceramides have only one acylgroup which can be readily varied in terms of its chain length anddegree of saturation. Ceramides suitable for use in accordance with thepresent invention are commercially available. In addition, ceramides canbe isolated, for example, from egg or brain using well-known isolationtechniques or, alternatively, they can be synthesized using the methodsand techniques disclosed in U.S. patent application Ser. No. 08/316,429,filed Sep. 30, 1994, and U.S. patent application Ser. No. 08/486,214,filed Jun. 7, 1995, the teachings of which are incorporated herein byreference. Using the synthetic routes set forth in the foregoingapplication, ceramides having saturated or unsaturated fatty acids withcarbon chain lengths in the range of C₂ to C₃₁ can be prepared.

[0099] In addition to the foregoing, detergents, proteins and peptidescan be used as bilayer stabilizing components. Detergents which can beused as bilayer stabilizing components include, but are not limited to,Triton X-100, deoxycholate, octylglucoside and lyso-phosphatidylcholine.Proteins which can be used as bilayer stabilizing components include,but are not limited to, glycophorin and cytochrome oxidase. Cleavage ofthe protein, by endogenous proteases, resulting in the loss of the bulkydomain external to the bilayer would be expected to reduce the bilayerstabilizing ability of the protein. In addition, peptides which can beused as bilayer stabilizing components include, for example, thepentadecapeptide, alanine-(aminobutyric acid-alanine)₁₄. This peptidecan be coupled, for example, to polyethyleneglycol which would promoteits transfer out of the bilayer. Alternatively, peptides such ascardiotoxin and melittin, both of which are known to induce non-lamellarphases in bilayers, can be coupled to PEG and might thereby be convertedto bilayer stabilizers in much the same way that PE is converted from anon-lamellar phase preferring lipid to a bilayer stabilizer when it iscoupled to PEG. If the bond between the peptide and the PEG is labile,then cleavage of the bond would result in the loss of the bilayerstabilizing ability and in the restoration of a non-lamellar phase,thereby causing the liposome to become fusogenic.

[0100] Typically, the bilayer stabilizing component is present at aconcentration ranging from about 0.05 mole percent to about 50 molepercent. In a presently preferred embodiment, the bilayer stabilizingcomponent is present at a concentration ranging from 0.05 mole percentto about 25 mole percent. In an even more preferred embodiment, thebilayer stabilizing component is present at a concentration ranging from0.05 mole percent to about 15 mole percent. One of ordinary skill in theart will appreciate that the concentration of the bilayer stabilizingcomponent can be varied depending on the bilayer stabilizing componentemployed and the rate at which the liposome is to become fusogenic.

[0101] By controlling the composition and concentration of the bilayerstabilizing component, one can control the rate at which the bilayerstabilizing component exchanges out of the liposome and, in turn, therate at which the liposome becomes fusogenic. For instance, when apolyethyleneglycol-phosphatidylethanolamine conjugate or apolyethyleneglycol-ceramide conjugate is used as the bilayer stabilizingcomponent, the rate at which the liposome becomes fusogenic can bevaried, for example, by varying the concentration of the bilayerstabilizing component, by varying the molecular weight of thepolyethyleneglycol, or by varying the chain length and degree ofsaturation of the acyl chain groups on the phosphatidylethanolamine orthe ceramide. In addition, other variables including, for example, pH,temperature, ionic strength, etc. can be used to vary and/or control therate at which the liposome becomes fusogenic. Other methods which can beused to control the rate at which the liposome becomes fusogenic willbecome apparent to those of skill in the art upon reading thisdisclosure.

[0102] In a presently preferred embodiment, the fusogenic liposomescontain cholesterol. It has been determined that when cholesterol-freeliposomes are used in vivo, they have a tendency to absorb cholesterolfrom plasma lipoproteins and cell membranes. Since this absorption ofcholesterol could, in theory, change the fusogenic behavior of theliposomes, cholesterol can be included in the fusogenic liposomes of thepresent invention so that little or no net transfer of cholesteroloccurs in vivo. Cholesterol, if included, is generally present at aconcentration ranging from 0.02 mole percent to about 50 mole percentand, more preferably, at a concentration ranging from about 35 molepercent to about 45 mole percent.

[0103] III. Fusogenic Liposomes Containing Fusogenic Lipopeptides

[0104] In another embodiment, the present invention provides fusogenicliposome containing a fusogenic lipopeptide. More particularly, thepresent invention provides a lipopetide, the lipopeptide comprising alipid covalently attached to a peptide by means of an amide bond. Onceformed, the lipopeptide can be incorporated into the outer monolayer ofa liposome or, alternatively, into both the inner and outer monolayersof a liposome. It has been discovered that the lipopeptide of thepresent invention form stable bilayers with numerous lipids at a higherpH (e.g., at a pH of about 7.5), but destabilization of these lipidvesicles can be induced by decreasing the pH (e.g., to a pH below about6.0). This membrane destabilization not only results in extensiveleakage of liposomal contents, but also in lipid mixing. Thus, when thelipopeptides of the present invention are incorporated into a liposome,the fusogenic properties of the liposome are enhanced.

[0105] As noted above, the lipopeptide of the present invention isformed by covalently attaching a lipid to a peptide by means of an amidebond. A variety of lipids can be used to form the lipopeptides of thepresent invention. In a presently preferred embodiment, a diacylglycerolis the lipid used to form the lipopeptide. Diacylglycerols suitable foruse in accordance with the present invention can have a variety of acylchain groups of varying chain lengths and degrees of saturation. Suchdiacylglycerols are commercially available, or can be isolated orsynthesized using conventional techniques known to those of skill in theart. Diacylglycerols containing saturated or unsaturated fatty acidswith carbon chain lengths in the range of C₁₀ to C₂₀ are preferred.Examples of such diacylglycerols include, but are not limited to,1,2-distearoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol,1,2-dipalmitoyl-sn-glycerol.

[0106] In addition, the peptide used to form the lipopeptide can be anypeptide know to promote membrane fusion, i.e., any fusogenicpH-sensitive peptides. Generally, such fusogenic peptides are short inlength, capable of insertion in a monolayer in order to destabilize thebilayer membrane and, in addition, such fusogenic peptides generallyhave a pH-induced conformational change creating well separatedhydrophilic and hydrophobic faces. Such fusogenic peptides can bederived from known viral fusion proteins, e.g., the viral fusion proteinof influenza hemagglutinin (HA). These peptides adopt amphipathicα-helical structures and penetrate lipid membranes at the pHcorresponding to the fusion of the native virus. Examples of fusogenicpeptides suitable for use in the lipopeptides of the present inventioninclude, but are not limited to, those described by S. Takahashi(“Conformation of Membrane Fusion-Active 20-Residue Peptides With orWithout Lipid Bilayers. Implication of α-Helix Formation for MembraneFusion,” Biochemistry, 29:6257-6264 (1990)); R. Ishiguro, et al.(“Orientation of Fusion-Active Synthetic Peptides in PhospholipidBilayers: Determination by Fourier Transform Infrared Spectroscopy,”Biochemistry, 32:9792-9797 (1993)); R. Brasseur, et al. (“Orientationinto the lipid bilayer of an asymmetric amphipathic helical peptidelocated at the N-terminus of viral fusion proteins,” Biochimica etBiophysica Acta, 1029:267-273 (1990)); S. Lee, et al., (“Effect ofAmphipathic peptides with different a-helical contents on liposomefusion,” Biochimica et Biophysica Acta, 1103:157-162 (1992)); K. Kono,et al., (“Fusion activity of an amphophilic polypeptide having acidicamino acid residues: generation of fusion activity by α-Helix formationand charge neutralization, ” Biochimica et Biophysica Acta, 1164:81-90(1993)); S. E. Glushakova, et al. (“The fusion of artificial lipidmembranes induced by the synthetic arenavirus ‘fusion peptide’,”Biochimica et Biophysica Acta, 1110:202-208 (1992)); M. Murata, et al.,“Specificity of Amphilic anionic peptides for fusion of phospholipidvesicles,” Biophysical Journal, 64:724-734 (1993); C. Puyal, et al.,“Design of a short membrane-destabilizing peptide covalently bound toliposomes,” Biochimica et Biophysica Acta, 1195:259-266 (1994); andGoormaghtigh, et al. (“Secondary structure and orientation of theamphipathic peptide GALA in lipid structures (An infrared-spectroscopicapproach),” European Journal of Biochemistry, 195:421-429 (1991)), theteachings of which are incorporated herein by reference for allpurposes.

[0107] More particularly, examples of fusogenic peptides suitable foruse in accordance with the present invention include, but are notlimited to, the following: Ac-GLFEAIAGFIENGWEGMIDGK (AcE4K);WEAALAEALAEALAEIILAEALAEALEALAA (GALA); GGYCLTRWMLIEAELKCFGNTAV (Lassa);GGYCLTKWMILAAELKCFGNTAV (LCM); GGYCLEKWMIVASELKCFGNTAI (Takaribe);GGYCLEQWAIIWAGIXCFDNTVM (Picinde); GLFEALAEFIEGGWEGLIEG (E5);GLFEAIAEFIEAIAEFIEG (E5NN); GWEGLIEGIEGGWEGLIEG (E5CC);GLFEAIAEFIPGGWEGLIEG (E5P); GLLEELLELLEELWEELLEG (E8);Ac-LARLLARLLARL-NHCH₃; Ac-LARLLPRLLARL-NHCH₃; Ac-LPRLLPRLLARL-NHCH₃;Ac-LPRLLPRLLPRL-NHCH₃; FEAALAEALAEALA (SFP-1); Myr-FEAAKAEAKAEAKA(SFP-2); WEAALAEALAEALAC (SFP-3); and poly(Glu-Aib-Leu-Aib)

[0108] wherein “Myr” is used to represent myristic acid and “Aib” isused to represent 2-aminoisobutyric acid. Other fusogenic peptides whichcan be used to form the lipopeptides of the present invention will beknown to those of skill in the art.

[0109] In addition, it will be readily apparent to those of ordinaryskill in the art that the fusogenic peptides set forth above can besubject to various changes, such as insertions, deletions, andsubstitutions, either conservative or nonconservative, where suchchanges might provide for certain advantages in their use, i.e., toincrease their fusogenic activity. By “conservative substitutions” ismeant replacing an amino acid residue with another which is biologicallyand/or chemically similar, e.g., one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Ieu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr. Residues which can be modified withoutloosing the biological activity of the fusogenic peptides can beidentified by single amino acid substitutions, deletions, or insertionsusing conventional techniques known to those of ordinary skill in theart, this is especially true of the peptides of the present inventionbeing that they are relatively short in length. In addition, thecontributions made by the side chains of the residues can be probed viaa systematic scan with a specified amino acid (e.g., Ala). As such,examples of fusogenic peptides which can be used to form thelipopeptides of the present invention include those set forth above andconservative modifications thereof.

[0110] The lipopeptide of the present invention is formed by covalentlyattaching a lipid to a peptide by means of an amide bond. The amide bondis generally formed between a carboxyl group of the lipid and an aminogroup, preferably a primary amino group, of the peptide. The amino groupcan be present at the N-terminus of the peptide or, alternatively, itcan be present in the side chain of the amino acid present at theC-terminus. It will understood by those of skill in the art that theamino groups which are not used to form the amide bond will be protectedusing standard techniques to prevent reactivity (see, below forillustrative examples of protecting groups for amino groups). In apreferred embodiment, the amino group used in the formation of the amidebond is present in a lysine residue at the C-terminus of the peptide. Ifa lysine residue is not naturally present at the C-terminus of thepeptide, one can readily be added using standard techniques known tothose of skill in the art. It has been determined that a lysine residuecan be added to the peptide and, in turn, used to form an amide bondwith the lipid without adversely affecting the biological properties ofthe peptide.

[0111]FIG. 40 illustrates a synthetic scheme which can be used to formthe lipopeptides of the present invention. This scheme illustrates theformation of a lipopeptide from the lipid 1,2-distearoyl-sn-glycerol andthe fusogenic peptide AcE4K, supra, using an amidation reaction. Thoseof skill in the art will readily appreciate that this synthetic schemeis illustrative and, thus, that it can be modified in numerous ways. Inthis reaction, one gram of 1,2-distearoyl-sn-glycerol (1.6 mmol) (1),0.2 g succinic anhydride (2 mmol), and 0.24 g 4-dimethylaminopyridine (2mmol) are dissolved in 10 ml of CH₂Cl₂ and stirred at room temperaturefor one hour. The resulting acid (2) is isolated by removing solvent byrotary evaporation followed by purification by silica gel chromatographyusing 10% ethyl acetate in hexane as eluant. About two hundredmilligrams of this material (0.28 mmol) and 32 mg ofN-hydroxysuccinimide (0.29 mmol) are dissolved in 5 ml of CH₂Cl₂ and 57mg of 1,3-dicyclohexylcarbodimide (0.28 mmol) is added with stirring.The reaction is allowed to proceed for about one hour at roomtemperature after which the mixture is filtered to remove precipitate,and the solvent is removed by rotary evaporation yielding the activatedlipid (3). A mixture of 5.6 mg of the peptide AcE4K (2.5 μmol), 4.1 mgof 3 (5.0 μmol) and 15 mg of triethylamine in 1 ml of dimethylsulfoxide(DMSO) are heated to 65° C. to achieve co-dissolution of the lipid andpeptide and incubated for one hour. After cooling, the lipopeptide (4)is precipitated by the addition of 5 ml of diethyl ether and centrifugedat 2000 rpm for 5 minutes. The pellet is washed three times with 2 ml ofdiethyl ether repeating the centrifugation with each wash. Thelipopeptide is dried under vacuum and its identity is confirmed by massspectrometry.

[0112] Once formed, the lipopeptide can be incorporated into the outermonolayer of a liposome or, alternatively, into both the inner and outermonolayers of a liposome. This is in contrast to the lipopeptideconjugates of the prior art which, as a result of the chemistry used tosynthesize them, can only be present on the outer monolayer of theliposome. If the lipopeptide of the present invention is to beincorporated only into the outer monolayer of the liposome, it is addedto a pre-formed liposome. Alternatively, if the lipopeptide of thepresent invention is to be incorporated into both the inner and outermonolayers of the liposome, then the lipopeptide is used as a componentin the formation of the liposome. The lipopeptides of the presentinvention can be used with liposomes prepared from a variety of lipids.In a preferred embodiment, the lipids used to prepare the liposomescontaining the lipopeptides are phosphoglycerides and, in particular,phosphatidylcholine. Examples of such phosphoglycerides are set forthabove. In a presently preferred embodiment, the lipids used to form theliposomes are 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC) and eggphosphatidylcholine (EPC). In a presently preferred embodiment, theliposomes also contain cholesterol.

[0113] Typically, the lipopeptide is present at a concentration rangingfrom about 0.05 mole percent to about 50 mole percent. In a presentlypreferred embodiment, the lipopeptide is present at a concentrationranging from 0.05 mole percent to about 25 mole percent. In an even morepreferred embodiment, the lipopeptide is present at a concentrationranging from 0.05 mole percent to about 10 mole percent. One of ordinaryskill in the art will appreciate that the concentration of thelipopeptide can be varied depending on the particular lipopeptideemployed and the rate at which the liposome is to become fusogenic.Cholesterol, if included, is generally present at a concentrationranging from 0.2 mole percent to about 50 mole percent and, morepreferably, at a concentration ranging from about 20 mole percent toabout 45 mole percent.

[0114] The lipopeptides of the present invention have significantadvantages over those in the prior art. As mentioned, in previously usedmethods, the fusion peptides are synthesized and anchored to lipidbilayers by a C-terminal cysteine linked to a bifunctionalphosphatidylethanolamine derivative (Puyal, et al., Biochimica etBiophysica Acta, 1195:259-266 (1994)). Unfortunately, there are a numberof drawbacks associated with the cysteine-thioether chemistry used byPuyal, et al. First, it is often difficult to prepare enough peptidecontaining a cystine residue at the C-terminus. Because they readilydimerize at a pH above 2, such proteins are difficult to synthesize andpurify. Second, it is difficult to control the composition of theendproduct. Again, the lipopeptide is prepared in situ and, thus, thereis a strong tendency for the peptide to dimerize. As a result of thedimerization of the peptide, it is difficult to achieve 100% reactionand, thus, to know what is truly present. Third, as a result of thechemistry used, leakage of the liposomal content often results. Fourth,the peptide can only be attached to the outer monolayer of a pre-formedliposome.

[0115] In contrast, the lipopeptides of the present invention can bereadily synthesized and purified. Because the chemistry used to preparethe lipopeptides of the present invention does not require a cysteineresidue to be present at the C-tenninus, the peptides do not dimerize.Moreover, with the lipopeptides of the present invention, one starts offwith a defined and purified product and, thus, one can control thesystem better, i.e., what one puts into the system, one gets back out.In addition, in contrast to the system of Puyal, et al., one canmanipulate the placement of the lipopeptides of the present invention.As previously mentioned, the lipopeptides of the present invention canbe incorporated into the outer monolayer of a liposome or,alternatively, into both the inner and outer monolayers of a liposome.In terms of increasing the fusogenic properties of a liposome, there aresignificant advantages to having the lipopeptide present in bothmonolayers of the liposome.

[0116] It should be noted that since the fusogenic peptides used in thelipopeptides of the present invention are relatively short in length,they can be prepared using any of a number of chemical peptide synthesistechniques well known to those of ordinary skill in the art, includingboth solution methods and solid phase methods, with solid phasesynthesis being presently preferred.

[0117] In particular, solid phase synthesis in which the C-terminalamino acid of the peptide sequence is attached to an insoluble supportfollowed by sequential addition of the remaining amino acids in thesequence is the preferred method for preparing the fusogenic peptides ofthe present invention. Techniques for solid phase synthesis aredescribed by Barany and Merrifield, Solid-Phase Peptide Synthesis, inThe Peptides: Analysis, Synthesis, Biology (Gross and Meienhofer (eds.),Academic Press, N.Y., vol. 2, pp. 3-284 (1980)); Merrifield, et al., J.Am. Chem. Soc., 85:2149-2156 (1963); and Stewart, et al., Solid PhasePeptide Synthesis (2nd ed., Pierce Chem. Co., Rockford, Ill. (1984)),the teachings of which are hereby incorporated by reference.

[0118] Solid phase synthesis is started from the carboxy-terminal end(i.e., the C-terminus) of the peptide by coupling a protected amino acidvia its carboxyl group to a suitable solid support. The solid supportused is not a critical feature of the present invention provided that itis capable of binding to the carboxyl group while remainingsubstantially inert to the reagents utilized in the peptide synthesisprocedure. For example, a starting material can be prepared by attachingan amino-protected amino acid via a benzyl ester linkage to achloromethylated resin or a hydroxymethyl resin or via an amide bond toa benzhydrylamine (BHA) resin or p-methylbenzhydrylamine (MBHA) resin.Materials suitable for us as solid supports are well known to those ofskill in the art and include, but are not limited to, the following:halomethyl resins, such as chloromethyl resin or bromomethyl resin;hydroxymethyl resins; phenol resins, such as4-(α-[2,4-dimethoxyphenyl]-Fmoc-aminomethyl)phenoxy resin;tert-alkyloxycarbonyl-hydrazidated resins, and the like. Such resins arecommercially available and their methods of preparation are known bythose of ordinary skill in the art.

[0119] The acid form of the peptides of the present invention may beprepared by the solid phase peptide synthesis procedure using a benzylester resin as a solid support. The corresponding amides may be producedby using benzhydrylamine or methylbenz-hydrylamine resin as the solidsupport. Those skilled in the art will recognize that when the BHA orMBHA resin is used, treatment with anhydrous hydrofluoric acid to cleavethe polypeptide from the solid support produces a polypeptide having aterminal amide group.

[0120] The α-amino group of each amino acid used in the synthesis shouldbe protected during the coupling reaction to prevent side reactionsinvolving the reactive α-amino function. Certain amino acids alsocontain reactive side-chain functional groups (e.g., sulfhydryl, amino,carboxyl, hydroxyl, etc.) which must also be protected with appropriateprotecting groups to prevent chemical reactions from occurring at thosesites during the polypeptide synthesis. Protecting groups are well knownto those of skill in the art. See, for example, The Peptides: Analysis,Synthesis, Biology, Vol. 3: Protection of Functional Groups in PeptideSynthesis (Gross and Meienhofer (eds.), Academic Press, N.Y. (1981)),the teachings of which are incorporated herein by reference.

[0121] A properly selected α-amino protecting group will render theα-amino function inert during the coupling reaction, will be readilyremovable after coupling under conditions that will not remove sidechain protecting groups, will not alter the structure of the peptidefragment, and will prevent racemization upon activation immediatelyprior to coupling. Similarly, side-chain protecting groups must bechosen to render the side chain functional group inert during thesynthesis, must be stable under the conditions used to remove theα-amino protecting group, and must be removable after completion of thepolypeptide synthesis under conditions that will not alter the structureof the polypeptide.

[0122] Illustrative examples of protecting groups for an α-amino groupinclude, but are not limited to, the following: aromatic urethane-typegroups such as, for example, fluorenylmethyloxycarbonyl (Fmoc),carbobenzoxy (Cbz), and substituted benzyloxycarbonyls includingp-chlorobenzyloxycarbonyl, o-chlorobenzyloxycarbonyl,2,4-dichlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl, etc.;aliphatic urethane-type groups such as, for example, butyloxycarbonyl(Boc), t-amyloxycarbonyl, isopropyloxycarbonyl,2-(p-biphenylyl)-isopropyloxycarbonyl, allyloxycarbonyl, etc.; andcycloalkyl urethane-type groups such as, for example,cyclopentyloxycarbonyl, cyclohexyloxycarbonyl, cycloheptyloxy-carbonyl,adamantyloxycarbonyl (Adoc), etc. In a presently preferred embodiment,fluorenylmethyloxycarbonyl (Fmoc) is the α-amino protecting group used.

[0123] For the side chain amino group present in lysine (Lys), any ofthe protecting groups described above for the protection of the α-aminogroup are suitable. Moreover, other suitable protecting groups include,but are not limited to, the following: butyloxycarbonyl (Boc),p-chlorobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,o-chlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl,2,4-dichlorobenzyl-oxycarbonyl, o-bromobenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, t-butyloxycarbonyl, isopropyloxycarbonyl,t-amyloxycarbonyl, cyclopentyloxycarbonyl, cyclohexyl-oxycarbonyl,cycloheptyloxycarbonyl, adamantyloxycarbonyl, p-toluenesulfonyl, etc. Ina presently preferred embodiment, the side chain amino protecting groupfor Lys is butyloxycarbonyl (Boc).

[0124] For protection of the guanidino group of arginine (Arg), examplesof suitable protecting groups include, but are not limited to, thefollowing: nitro, tosyl (Tos), carbobenzoxy (Cbz), adamantyloxycarbonyl(Adoc), butyloxycarbonyl (Boc), 4-methoxy-2, 3,6-trimethylbenzenesulfonyl (Mtr) and 2,2,5,7,8-pentamethylchloroman-6-sulfonyl (PMC). In a presently preferredembodiment, 4-methoxy-2,3,6-trimethyl-benzenesulfonyl and2,2,5,7,8-pentamethylchloroman-6-sulfonyl are the protecting group usedfor Arg.

[0125] The hydroxyl group on the side chains of serine (Ser), threonine(Thr) or tyrosine (Tyr) can be protected by a C₁-C₄ alkyl such as, forexample, methyl, ethyl and t-butyl, or by a substituted benzyl such as,for example, p-methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl,o-chlorobenzyl and 2,6-dichlorobenzyl. The preferred aliphatic hydroxylprotecting group for Ser, Thr and Tyr is t-butyl.

[0126] The carboxyl group of aspartic acid (Asp) may be protected by,for example, esterification using groups such as benzyl, t-butyl,cyclohexyl, cyclopentyl, and the like. For Asp, t-butyl is the presentlypreferred protecting group.

[0127] The basic imidazole ring in histidine (His) may be protected by,for example, t-butoxymethyl (Bom), butyloxycarbonyl (Boc) andfluorenylmethyloxycarbonyl (Fmoc). In a preferred embodiment,t-butoxymethyl (Bom) is the protecting group used.

[0128] Coupling of the amino acids may be accomplished by a variety ofchemistries known to those of skill in the art. Typical approachesinvolve either the conversion of the amino acid to a derivative thatwill render the carboxyl group more susceptible to reaction with thefree N-terminal amino group of the polypeptide fragment, or use of asuitable coupling agent such as, for example,N,N′-dicyclohexylcarbodimide (DCC) or N,N′-diisopropylcarbodiimide(DIPCDI). Frequently, hydroxybenzotriazole (HOBt) is employed as acatalyst in these coupling reactions. Appropriate synthesis chemistriesare disclosed in The Peptides: Analysis, Structure, Biology, Vol. 1:Methods of Peptide Bond Formation (Gross and Meienhofer (eds.), AcademicPress, N.Y. (1979)); and Izumiya, et al., Synthesis of Peptides (MaruzenPublishing Co., Ltd., (1975)), both of which are incorporated herein byreference.

[0129] Generally, synthesis of the polypeptide is commenced by firstcoupling the C-terminal amino acid, which is protected at the Nα-aminoposition by a protecting group such as fluorenylmethyloxycarbonyl(Fmoc), to a solid support. Prior to coupling of Fmoc-Asn, the Fmocresidue has to be removed from the polymer. Fmoc-Asn can, for example,be coupled to the 4-(α-[2,4-dimethoxyphenyl]-Fmoc-amino-methyl)phenoxyresin using N,N′-dicyclohexylcarbodimide (DCC) and hydroxybenzotriazole(HOBt) at about 25° C. for about two hours with stirring. Following thecoupling of the Fmoc-protected amino acid to the resin support, theα-amino protecting group is removed using 20% piperidine in DMF at roomtemperature.

[0130] After removal of the α-amino protecting group, the remainingFmoc-protected amino acids are coupled stepwise in the desired order.Appropriately protected amino acids are commercially available from anumber of suppliers (e.g., Nova (Switzerland) or Bachem (California)).As an alternative to the stepwise addition of individual amino acids,appropriately protected peptide fragments consisting of more than oneamino acid may also be coupled to the “growing” polypeptide. Selectionof an appropriate coupling reagent, as explained above, is well known tothose of skill in the art. It should be noted that since the fusogenicpeptides of the present invention are relative short in length, thislatter approach (i.e., the segment condensation method) is not the mostefficient method of peptide synthesis.

[0131] Each protected amino acid or amino acid sequence is introducedinto the solid phase reactor in excess and the coupling is carried outin a medium of dimethylformamide (DMF), methylene chloride (CH₂Cl₂) or,mixtures thereof. If coupling is incomplete, the coupling reaction maybe repeated before deprotection of the Nα-amino group and addition ofthe next amino acid. Coupling efficiency may be monitored by a number ofmeans well known to those of skill in the art. A preferred method ofmonitoring coupling efficiency is by the ninhydrin reaction. Polypeptidesynthesis reactions may be performed automatically using a number ofcommercially available peptide synthesizers (e.g., Biosearch 9500,Biosearch, San Raphael, Calif.).

[0132] The peptide can be cleaved and the protecting groups removed bystirring the insoluble carrier or solid support in anhydrous, liquidhydrogen fluoride (HF) in the presence of anisole and dimethylsulfide atabout 0° C. for about 20 to 90 minutes, preferably 60 minutes; bybubbling hydrogen bromide (HEBr) continuously through a 1 mg/10 mLsuspension of the resin in trifluoroacetic acid (TFA) for 60 to 360minutes at about room temperature, depending on the protecting groupsselected; or, by incubating the solid support inside the reaction columnused for the solid phase synthesis with 90% trifluoroacetic acid, 5%water and 5% triethylsilane for about 30 to 60 minutes. Otherdeprotection methods well known to those of skill in the art may also beused.

[0133] The fusogenic peptides can be isolated and purified from thereaction mixture by means of peptide purification well known to those ofskill in the art. For example, the polypeptides may be purified usingknown chromatographic procedures such as reverse phase HPLC, gelpermeation, ion exchange, size exclusion, affinity, partition, orcountercurrent distribution.

[0134] Although the fusogenic peptides are preferably synthesized orprepared using chemical peptide synthesis techniques such as describedabove, it will be understood by those of ordinary skill in the art thatthey can also be prepared by other means including, for example,recombinant techniques. Two text books which describe suitablerecombinant techniques in great detail are Sambrook, et al., MolecularCloning, A Laboratory Manual (Cold Spring Harbor Publish., Cold SpringHarbor, N.Y. 2nd ed. (1989)), Methods in Enzymology, Vol. 152: Guide toMolecular Cloning Techniques (Berger and Kimmel (eds.), San Diego:Academic Press, Inc. (1987)) and Kriegler, Gene Transfer and Expression:A Laboratory Manual (W. H. Freeman, N.Y. (1990)), the teachings of whichare incorporated herein by reference.

[0135] IV. Fusogenic Liposomes Containing Fusogenic Polymers

[0136] In another embodiment, the present invention provides apH-sensitive fusogenic oligomer or, alternatively, a pH-sensitivefusogenic polymer. More particularly, the present invention provides apolymer having the general structure:

[X—Y]_(n)

[0137] in which: X is a trifunctional substrate wherein at least one ofthe functional groups is a carboxyl group or a protected carboxyl group;Y is ethylene glycol; and “n” is an integer having a value ranging from1 to about 30, more preferably, from 1 to about 20 and, even morepreferably, from 2 to about 10.

[0138] A “trifunctional substrate,” as used herein, refers to a compoundthat contains three functional groups, at least one of which is acarboxy group or a protected carboxyl group as it is the carboxylgroup(s) which imparts pH-sensitivity to the polymer. Examples oftrifunctional substrates suitable for use in accordance with the presentinvention include, but are not limited to, compounds containing at leastone carboxyl group and, in addition, one or more of the following: anamino group, a hydroxy group, a ketone, an aldehyde, a thiol, afunctional group which allows for further chain extension orderivatization, or a combination of these various functional groups. Ina presently preferred embodiment, L-glutamic acid is the trifunctionalsubstrate used.

[0139] “Ethylene glycol,” as used herein, generally refers to acompounds having the formula: (CH₂OH.CH₂OH)_(n), wherein n has a valueranging from 1 to about 8. Examples of ethylene glycols which aresuitable for use in accordance with the present invention include, butare not limited to, di(ethylene glycol), tri(ethylene glycol),tetra(ethylene glycol), penta(ethylene glycol), hexa(ethylene glycol),octa(ethylene glycol) etc. Such ethylene glycols are commerciallyavailable from a number of different sources including, for example,Aldrich Chemical Co. (Milwaukee, Wis.). In a presently preferredembodiment, tetraethylene glycol (TEG) is the ethylene glycol used.

[0140] In another embodiment, the present invention provides a polymerhaving the general structure:

[X—Y—Z]_(n)

[0141] in which: X and Z are independently selected and aretrifunctional substrates wherein at least one of the functional groupsis a carboxyl group or a protected carboxyl group; Y is an ethyleneglycol; and “n” is an integer having a value ranging from 1 to about 30,more preferably, from 1 to about 20 and, even more preferably, from 2 toabout 10. The term “independently selected” is used herein to indicatethat the trifunctional substrates, i.e., X and Z, may be identical ordifferent (e.g., X and Y may both be L-glutamic acid, etc.) Thetrifunctional substrate and the ethylene glycol used to form the abovepH-sensitive fusogenic oligomer are as defined above.

[0142]FIGS. 51 through 53 illustrate the synthetic schemes which can beused to form the linkage of different combinations of a trifunctionalsubstrate (e.g., L-glutamic acid (Glu)) and a short chainpolyoxyethylene of uniform size (e.g., tetraethylene glycol (TEG)) toform a linear chain of defined length. FIG. 51 illustrates the reactionsteps used to prepare the basic units for synthesizing glutamicacid-tetraethylene glycol oligomers. Tetraethylene glycol (TEG) iscondensed with N-t-Boc-τ-Bz-L-glutamic acid by dicyclohexylcarbodiimide(DCC)/4-dimethylaminopyridine (DMAP) to form the first basic buildingunit: t-Boc-BzGlu-TEG (1). Activation of the latter with excessdi(N-succinimidyl) carbonate (DSC) in the presence of DMAP (Step 2)enables the subsequent conjugation with BzGlu (Step 3) to form thesecond basic unit: t-Boc-Bz-Glu-TEG-BzGlu (2).

[0143] As shown in FIG. 52, the first basic monomeric unit (1) can beused to generate the oligomer with the structure [Glu-TEG]_(n) (n=anynumber), whereas the second basic unit (2) can be used to generate theoligomer with the structure [Glu-TEG-Glu]_(n) (n=any number) shown inFIG. 53. It should be noted that in both sequences, doubling of thechain length can be performed in one reaction and this can be repeatedaccording to the final size of the oligomer required in order to providethe desired properties and characteristic when applied to thetransmembrane carrier system. Therefore, the number of steps to obtain along chain is reduced significantly. Furthermore, the fusogenicoligomers of the present invention are designed such that they can bereadily conjugated to a lipid anchor at one terminal end and/or to atargeting ligand or other factor at the other terminal.

[0144] Once formed, the pH-sensitive fusogenic polymers of the presentinvention can be incorporated into or covalently attached to liposomevesicles, lipid particles or other lipid carrier systems using methodsknown to and used by those of skill in the art. Lipids which can be usedto form the lipid carrier systems containing the pH-sensitive fusogenicoligomers of the present invention include phosglycerides andsphingolipids. Representative examples of suitable phosglycerides andsphingolipids are set forth in the Glossary Section, supra.

[0145] The pH-sensitive fusogenic polymers of the present inventiontrigger fusion or release of the contents of the carrier system onprotonation of the carboxyl groups when the carrier system encounters anacidic environment. One of the advantages of the fusogenic polymers ofthe present invention is that they are of defined chain-lengths and,thus, one can readily control their pH-sensitivity. This is in contrastto previously used polymers which usually contain a mixture of differentchain-lengths. As a result of the varying chain-lengths, the number ofcarboxyl groups introduced often varies between different preparationsand, unfortunately, such variance causes fluctuation in the propertiesof the resulting systems and inconsistency in the product. Again, incontrast to such polymers, the pH-sensitive fusogenic polymers of thepresent invention are of a defined size, i.e., a defined chain-length.

[0146] V. Methods of Preparing Liposomes

[0147] A variety of methods are available for preparing liposomes asdescribed in, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467(1980), U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975,4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,235,871, 4,261,975,4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,946,787, PCT PublicationNo. WO 91/17424, Deamer and Bangham, Biochim. Biophys. Acta, 443:629-634(1976); Fraley, et al., Proc. Natl. Acad. Sci. USA, 76:3348-3352 (1979);Hope, et al., Biochim. Biophys. Acta, 812:55-65 (1985); Mayer, et al.,Biochim. Biophys. Acta, 858:161-168 (1986); Williams, et al., Proc.Natl. Acad. Sci. USA, 85:242-246 (1988); the text Liposomes, (Marc J.Ostro (ed.), Marcel Dekker, Inc., New York, 1983, Chapter 1); and Hope,et al., Chem. Phys. Lip., 40:89 (1986), all of which are incorporatedherein by reference. Suitable methods include, for example, sonication,extrusion, high pressure/homogenization, microfluidization, detergentdialysis, calcium-induced fusion of small liposome vesicles andether-fusion methods, all of which are well known in the art. One methodproduces multilamellar vesicles of heterogeneous sizes. In this method,the vesicle-forming lipids are dissolved in a suitable organic solventor solvent system and dried under vacuum or an inert gas to form a thinlipid film. If desired, the film may be redissolved in a suitablesolvent, such as tertiary butanol, and then lyophilized to form a morehomogeneous lipid mixture which is in a more easily hydrated powder-likeform. This film is covered with an aqueous buffered solution and allowedto hydrate, typically over a 15-60 minute period with agitation. Thesize distribution of the resulting multilamellar vesicles can be shiftedtoward smaller sizes by hydrating the lipids under more vigorousagitation conditions or by adding solubilizing detergents such asdeoxycholate.

[0148] Unilamellar vesicles are generally prepared by sonication orextrusion. Sonication is generally preformed with a tip sonifier, suchas a Branson tip sonifier, in an ice bath. Typically, the suspension issubjected to several sonication cycles. Extrusion can be carried out bybiomembrane extruders, such as the Lipex Biomembrane Extruder. Definedpore size in the extrusion filters can generate unilamellar liposomalvesicles of specific sizes. The liposomes can also be formed byextrusion through an asymmetric ceramic filter, such as a CeraflowMicrofilter, commercially available from the Norton Company, WorcesterMass.

[0149] Following liposome preparation, the liposomes may be sized toachieve a desired size range and relatively narrow distribution ofliposome sizes. A size range of about 0.05 microns to about 0.20 micronsallows the liposome suspension to be sterilized by filtration through aconventional filter, typically a 0.22 micron filter. The filtersterilization method can be carried out on a high through-put basis ifthe liposomes have been sized down to about 0.05 microns to about 0.20microns.

[0150] Several techniques are available for sizing liposomes to adesired size. One sizing method is described in U.S. Pat. No. 4,737,323,incorporated herein by reference. Sonicating a liposome suspensioneither by bath or probe sonication produces a progressive size reductiondown to small unilamellar vesicles less than about 0.05 microns in size.Homogenization is another method which relies on shearing energy tofragment large liposomes into smaller ones. In a typical homogenizationprocedure, multilamellar vesicles are recirculated through a standardemulsion homogenizer until selected liposome sizes, typically betweenabout 0.1 and 0.5 microns, are observed. In both of these methods, theparticle size distribution can be monitored by conventional laser-beamparticle size discrimination. In addition, the size of the liposomalvesicle can be determined by quasi-electric light scattering (QELS) asdescribed in Bloomfield, Ann. Rev. Biophys. Bioeng. 10:421-450 (1981),incorporated herein by reference. Average liposome diameter can bereduced by sonication of formed liposomes. Intermittent sonicationcycles can be alternated with QELS assessment to guide efficientliposome synthesis.

[0151] Extrusion of liposome through a small-pore polycarbonate membraneor an asymmetric ceramic membrane is also an effective method forreducing liposome sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired liposome size distribution is achieved. Theliposomes may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in liposome size. For use in the presentinventions, liposomes having a size of from about 0.05 microns to about0.45 microns are preferred.

[0152] For the delivery of therapeutic agents, the fusogenic liposomesof the present invention can be loaded with a therapeutic agent andadministered to the subject requiring treatment. The therapeutic agentswhich can be administered using the fusogenic liposomes of the presentinvention can be any of a variety of drugs, peptides, proteins, DNA, RNAor other bioactive molecules. Moreover, cationic lipids may be used inthe delivery of therapeutic genes or oligonucleotides intended to induceor to block production of some protein within the cell. Nucleic acid isnegatively charged and must be combined with a positively charged entityto form a complex suitable for formulation and cellular delivery.

[0153] Cationic lipids have been used in the transfection of cells invitro and in vivo (Wang, C-Y, Huang L., “pH sensitive immunoliposomesmediate target cell-specific delivery and controlled expression of aforeign gene in mouse,” Proc. Natl. Acad. Sci. USA, 1987; 84:7851-7855and Hyde, S. C., Gill, D. R., Higgins, C. F., et al., “Correction of theion transport defect in cystic fibrosis transgenic mice by genetherapy,” Nature, 362:250-255 (1993)). The efficiency of thistransfection has often been less than desired, for various reasons. Oneis the tendency for cationic lipids complexed to nucleic acid to formunsatisfactory carriers. These carriers are improved by the addition ofPEG-modified lipids and, in particular, PEG-modified ceramide lipids.The addition of PEG-modified lipids prevents particle aggregation andprovides a means for increasing circulation lifetime and increasing thedelivery of the lipid-nucleic acid particles to the target cells.Moreover, it has been found that cationic lipids fuse more readily withthe target cells and, thus, the addition of neutrally chargedPEG-modified ceramide lipids does not mask or diminish the positivecharge of the carrier liposomes.

[0154] Cationic lipids useful in producing lipid based carriers for geneand oligonucleotide delivery include, but are not limited to,3β-(N-(N′,N′-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol);N,N-distearyl-N,N-dimethylammonium bromide (DDAB);N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE); diheptadecylamidoglycyl spermidine (DOGS);N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate (DOSPA);N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP);N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA);N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); LIPOFECTIN, acommercially available cationic lipid comprising DOTMA and DOPE(GIBCO/BRL, Grand Island, N.Y.) (U.S. Pat. Nos. 4,897,355; 4,946,787;and 5,208,036 issued to Epstein, et al.); LIPOFECTACE or DDAB(dimethyldioctadecyl ammonium bromide) (U.S. Pat. No. 5,279,883 issuedto Rose); LIPOFECTAMINE, a commercially available cationic lipidcomposed of DOSPA and DOPE (GIBCO/BRL, Grand Island, N.Y.); TRANSFECTAM,a commercially available cationic lipid comprising DOGS (Promega Corp.,Madison, Wis.).

[0155] Any variety of drugs which are selected to be an appropriatetreatment for the disease to be treated can be administered using thefusogenic liposomes of the present invention. Often the drug will be anantineoplastic agent, such as vincristine, doxorubicin, cisplatin,bleomycin, cyclophosphamide, methotrexate, streptozotocin, and the like.It may also be desirable to deliver anti-infective agents to specifictissues by the present methods. The compositions of the presentinvention can also be used for the selective delivery of other drugsincluding, but not limited to local anesthetics, e.g., dibucaine andchlorpromazine; beta-adrenergic blockers, e.g., propranolol, timolol andlabetolol; antihypertensive agents, e.g., clonidine and hydralazine;anti-depressants, e.g., imipramine, amitriptyline and doxepim;anti-convulsants, e.g., phenytoin; antihistamines, e.g.,diphenhydramine, chlorphenirimine and promethazine; antibacterialagents, e.g., gentamycin; antifungal agents, e.g., miconazole,terconazole, econazole, isoconazole, butaconazole, clotrimazole,itraconazole, nystatin, naftifine and amphotericin B; antiparasiticagents, hormones, hormone antagonists, immunomodulators,neurotransmitter antagonists, antiglaucoma agents, vitamins, narcotics,and imaging agents. Other particular drugs which can be selectivelyadministered by the compositions of the present invention will be wellknown to those of skill in the art. Additionally, two or moretherapeutic agents may be administered simultaneously if desired, wheresuch agents produce complementary or synergistic effects.

[0156] Methods of loading conventional drugs into liposomes include anencapsulation technique and the transmembrane potential loading method.In one encapsulation technique, the drug and liposome components aredissolved in an organic solvent in which all species are miscible andconcentrated to a dry film. A buffer is then added to the dried film andliposomes are formed having the drug incorporated into the vesiclewalls. Alternatively, the drug can be placed into a buffer and added toa dried film of only lipid components. In this manner, the drug willbecome encapsulated in the aqueous interior of the liposome. The bufferwhich is used in the formation of the liposomes can be any biologicallycompatible buffer solution of, for example, isotonic saline, phosphatebuffered saline, or other low ionic strength buffers. Generally, thedrug will be present in an amount of from about 0.01 ng/mL to about 50mg/mL. The resulting liposomes with the drug incorporated in the aqueousinterior or in the membrane are then optionally sized as describedabove.

[0157] Transmembrane potential loading has been described in detail inU.S. Pat. No. 4,885,172, U.S. Pat. No. 5,059,421, and U.S. Pat. No.5,171,578, the contents of which are incorporated herein by reference.Briefly, the transmembrane potential loading method can be used withessentially any conventional drug which exhibits weak acid or weak basecharacteristics. Preferably, the drug will be relatively lipophilic sothat it will partition into the liposome membrane. A pH gradient iscreated across the bilayers of the liposomes or protein-liposomecomplexes, and the drug is loaded into the liposome in response to thepH gradient. The pH gradient is generated by creating a proton gradientacross the membrane either by making the interior more acidic or basicthan the exterior (Harrigan, et al., Biochem. Biophys. Acta.,1149:329-339 (1993), the teachings of which are incorporated herein byreference), or by establishing an ion gradient employing ionizableagents, such as ammonium salts, which leads to the generation of a pHgradient (See, U.S. Pat. No. 5,316,771 (Barenholz), the teachings ofwhich are incorporated herein by reference).

[0158] In certain embodiments of the present invention, it is desirableto target the liposomes of the invention using targeting moieties thatare specific to a particular cell type, tissue, and the like. Targetingof liposomes using a variety of targeting moieties (e.g., ligands,receptors and monoclonal antibodies) has been previously described (see,e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044, both of which areincorporated herein by reference).

[0159] Examples of targeting moieties include monoclonal antibodiesspecific to antigens associated with neoplasms, such as prostate cancerspecific antigen. Tumors can also be diagnosed by detecting geneproducts resulting from the activation or overexpression of oncogenes,such as ras or c-erB2. In addition, many tumors express antigensnormally expressed by fetal tissue, such as the alphafetoprotein (AFP)and carcinoembryonic antigen (CEA). Sites of viral infection can bediagnosed using various viral antigens such as hepatitis B core andsurface antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Barr virusantigens, human immunodeficiency type-1 virus (HIV1) and papilloma virusantigens. Inflammation can be detected using molecules specificallyrecognized by surface molecules which are expressed at sites ofinflammation such as integrins (e.g., VCAM-1), selectin receptors (e.g.,ELAM-1) and the like.

[0160] Standard methods for coupling targeting agents to liposomes canbe used. These methods generally involve incorporation into liposomeslipid components, e.g., phosphatidylethanolamine, which can be activatedfor attachment of targeting agents, or derivatized lipophilic compounds,such as lipid derivatized bleomycin. Antibody targeted liposomes can beconstructed using, for instance, liposomes which incorporate protein A(see, Renneisen, et al., J. Biol. Chem., 265:16337-16342 (1990) andLeonetti, et al., Proc. Natl. Acad. Sci. (USA), 87:2448-2451 (1990),both of which are incorporated herein by reference).

[0161] Targeting mechanisms generally require that the targeting agentsbe positioned on the surface of the liposome in such a manner that thetarget moieties are available for interaction with the target, forexample, a cell surface receptor. The liposome is typically fashioned insuch a way that a connector portion is first incorporated into themembrane at the time of forming the membrane. The connector portion musthave a lipophilic portion which is firmly embedded and anchored in themembrane. It must also have a hydrophilic portion which is chemicallyavailable on the aqueous surface of the liposome. The hydrophilicportion is selected so that it will be chemically suitable to form astable chemical bond with the targeting agent which is added later.Therefore, the connector molecule must have both a lipophilic anchor anda hydrophilic reactive group suitable for reacting with the target agentand holding the target agent in its correct position, extended out fromthe liposome's surface. In some cases it is possible to attach thetarget agent to the connector molecule directly, but in most instancesit is more suitable to use a third molecule to act as a chemical bridge,thus linking the connector molecule which is in the membrane with thetarget agent which is extended, three dimensionally, off of the vesiclesurface.

[0162] Following a separation step as may be necessary to remove freedrug from the medium containing the liposome, the liposome suspension isbrought to a desired concentration in a pharmaceutically acceptablecarrier for administration to the patient or host cells. Manypharmaceutically acceptable carriers may be employed in the compositionsand methods of the present invention. Suitable formulations for use inthe present invention are found in Remington's Phannaceutical Sciences,Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). A varietyof aqueous carriers may be used, for example, water, buffered water,0.4% saline, 0.3% glycine, and the like, and may include glycoproteinsfor enhanced stability, such as albumin, lipoprotein, globulin, etc.Generally, normal buffered saline (135-150 mM NaCl) will be employed asthe pharmaceutically acceptable carrier, but other suitable carrierswill suffice. These compositions can be sterilized by conventionalliposomal sterilization techniques, such as filtration. The compositionsmay contain pharmaceutically acceptable auxiliary substances as requiredto approximate physiological conditions, such as pH adjusting andbuffering agents, tonicity adjusting agents, wetting agents and thelike, for example, sodium acetate, sodium lactate, sodium chloride,potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamine oleate, etc. These compositions can be sterilized usingthe techniques referred to above or, alternatively, they can be producedunder sterile conditions. The resulting aqueous solutions may bepackaged for use or filtered under aseptic conditions and lyophilized,the lyophilized preparation being combined with a sterile aqueoussolution prior to administration.

[0163] The concentration of liposomes in the carrier may vary.Generally, the concentration will be about 20-200 mg/ml, usually about50-150 mg/ml, and most usually about 75-125 mg/ml, e.g., about 100mg/ml. Persons of skill may vary these concentrations to optimizetreatment with different liposome components or for particular patients.For example, the concentration may be increased to lower the fluid loadassociated with treatment.

[0164] The present invention also provides methods for introducingtherapeutic compounds into cells of a host. The methods generallycomprise administering to the host a fusogenic liposome containing thetherapeutic compound. The host may be a variety of animals, includinghumans, non-human primates, avian species, equine species, bovinespecies, swine, lagomorpha, rodents, and the like.

[0165] The cells of the host are usually exposed to the liposomalpreparations of the invention by in vivo administration of theformulations, but ex vivo exposure of the cells to the liposomes is alsofeasible. In vivo exposure is obtained by administration of theliposomes to host. The liposomes may be administered in many ways. Theseinclude parenteral routes of administration, such as intravenous,intramuscular, subcutaneous, and intraarterial. Generally, the liposomeswill be administered intravenously or in some cases via inhalation.Often, the liposomes will be administered into a large central vein,such as the superior vena cava or inferior vena cava, to allow highlyconcentrated solutions to be administered into large volume and flowvessels. The liposomes may be administered intraarterially followingvascular procedures to deliver a high concentration directly to anaffected vessel. In some instances, the liposomes may be administeredorally or transdermally, although the advantages of the presentinvention are best realized by parenteral administration. The liposomesmay also be incorporated into implantable devices for long durationrelease following placement.

[0166] As described above, the liposomes will generally be administeredintravenously or via inhalation in the methods of the present invention.Often multiple treatments will be given to the patient. The dosageschedule of the treatments will be determined by the disease and thepatient's condition. Standard treatments with therapeutic compounds thatare well known in the art may serve as a guide to treatment withliposomes containing the therapeutic compounds. The duration andschedule of treatments may be varied by methods well known to those ofskill, but the increased circulation time and decreased in liposomeleakage will generally allow the dosages to be adjusted downward fromthose previously employed. The dose of liposomes of the presentinvention may vary depending on the clinical condition and size of theanimal or patient receiving treatment. The standard dose of thetherapeutic compound when not encapsulated may serve as a guide to thedose of the liposome-encapsulated compound. The dose will typically beconstant over the course of treatment, although in some cases the dosemay vary. Standard physiological parameters may be assessed duringtreatment that may be used to alter the dose of the liposomes of theinvention.

[0167] Liposome charge is an important determinant in liposome clearancefrom the blood, with negatively charged liposomes being taken up morerapidly by the reticuloendothelial system (Juliano, Biochem. Biophys.Res. Commun. 63:651(1975)) and, thus, having shorter half-lives in thebloodstream. Liposomes with prolonged circulation half-lives aretypically desirable for therapeutic and diagnostic uses. To maximizecirculation half-lives, the bilayer stabilizing component should be ahydrophilic polymer, e.g., PEG, conjugated to lipid anchors, e.g., PEs,having long, saturated hydrocarbon chains (C18>C16>C14) as theseconjugates provide a longer lasting steric barrier. As such, by varyingthe charge in addition to the foregoing factors, one of skill in the artcan regulate the rate at which the liposomes of the present inventionbecome fusogenic.

[0168] Additionally, the liposome suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as alphatocopherol, and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

[0169] It will be readily apparent to those of skill in the art thatvarious light sensitive, heat sensitive or pH-sensitive liposomes can beincorporated into the liposomes of the present invention to furtherenhance delivery of the therapeutic agent at the site of interest. Suchliposomes and methods for triggering the release of the contents of suchliposomes are described in U.S. Pat. No. 4,873,089, issued Oct. 10, 1989to Scotto, et al., entitled PROTEOLIPOSOMES AS DRUG CARRIERS; U.S. Pat.No. 4,882,165, issued Nov. 21, 1989 to Hunt, et al, entitled LIGHTSENSITIVE LIPOSOMES; and U.S. Pat. No. 4,801,459, issued Jan. 31, 1989to Liburdy, entitled TECHNIQUE FOR DRUG AND CHEMICAL DELIVERY, theteachings of which are incorporated herein by reference for allpurposes.

[0170] For instance, U.S. Pat. No. 5,277,913 discloses a triggeredrelease liposomal delivery system that selectively releases its contentsin response to illumination or reduction in pH. The liposomes contain anamphipathic lipid, such as a phospholipid, having two chains derivedfrom fatty acid that allow the lipid to pack into a bilayer structure.One or both of the alkyl chains contains a vinyl ether functionalitythat is cleaved by reactive oxygen species (ROS) or acid. Aphotosensitizer is incorporated into the liposomal cavity or membrane,and produces ROS or acid when illuminated to cleave the vinyl etherfunctionality and disrupt the liposomal membrane to release the vesiclecontents. The lipid is preferably a plasmalogen, for example

[0171] wherein R₁ and R₂ are each long chain hydrocarbons containing12-24 carbons; and R³ is a bilayer forming phosphoryl ester, such ascholine, ethanolamine, serine or inositol.

[0172] Another approach involves promoting leakage of liposome contentsby heating a liposomal saturated target site above a criticaltemperature range, for example, by radio frequency heating of targettissues. Yatvin, et al., Science, 202:1290 (1978). Another approach hasused liposomes prepared from pH-sensitive lipids, which leak theirpharmaceutical contents into low pH target regions. Such areas oflocalized acidity are sometimes found in tumors; hence, it has beenproposed that intravenous administration of such liposomes wouldpreferably selectively release anti-cancer chemotherapeutic agents attarget tumors (see, e.g., Yatvin, et al., Science, 210:1253 (1980)). ApH sensitive lipid is defined herein as a lipid that undergoes achemical or conformational change upon exposure to a decreased pH.

[0173] In addition, U.S. Pat. No. 4,882,165 similarly discloses alight-sensitive liposome which undergoes a trans to cis isomerizationupon irradiation with an appropriate wavelength of light (ultravioletlight) to allow the fluid contents of the liposome to escape through themembrane into the surrounding environment. Finally, GB Patent 2,209,468discloses liposomes having incorporated therein a photosensitizing agentthat absorbs light and alters the lipid membrane to release a drug fromthe liposome.

[0174] Such liposomes and triggering systems can advantageously be usedin combination with the liposomes of the present invention to furtherenhance their properties.

[0175] VI. Virosome-Mediated Intracellular Delivery of TherapeuticAgents

[0176] In another embodiment, the present invention provides virosomecompositions and methods for introducing a therapeutic compound intocells of a host. Liposomes having membrane-bound viral envelope fusionprotein (referred to herein as “virosomes”) are employed as carriers forthe therapeutic compounds. As explained in more detail below, the viralfusion protein facilitates membrane fusion between the virosome and cellmembranes to release the therapeutic compound into the cell cytoplasm.

[0177] “Liposome”, “vesicle” and “liposome vesicle” will be understoodto indicate structures having lipid-containing membranes enclosing anaqueous interior. The structures may have one or more lipid membranesunless otherwise indicated, although generally the liposomes will haveonly one membrane. Such single-layered liposomes are referred to hereinas “unilamellar”. Multilayer liposomes are referred to herein as“multilamellar”.

[0178] The virosomes present in the pharmaceutical compositions of thepresent invention have at least one viral fusion protein, such asinfluenza hemagglutinin, in the membranes of the liposomes. Thisstructure typically requires insertion of the viral fusion protein inthe liposome membrane during preparation, as generally described inBron, et al., Meth. Enzymol., 220:313-331 (1993) and Stegmann, et al.,EMBO J., 6:2651-2659 (1987), incorporated herein by reference. Thevirosomes can also be prepared from other viruses which have lipidbilayer envelopes, such as Semliki Forest virus containing the viralfusion protein E1-E2, vesicular stomatitis virus having the G protein asa membrane fusion protein, Sendai virus having the HN and F membranefusion proteins, and others.

[0179] For preparing virosomes, the viral membrane fusion protein suchas, e.g., hemagglutinin, is often purified from the corresponding virus,but it can also be produced by recombinant techniques. Purification ofhemagglutinin from viral stocks is described in more detail below.Hemagglutinin from human strains of influenza A, influenza B, orinfluenza C, or animal (avian, swine, equine, and the like) influenzastrains may be used to prepare the virosomes, although influenza Ahemagglutinin is generally preferred. A wide variety of suitable virusstocks are generally available as a hemagglutinin source, such as may beavailable from the American Type Culture Collection (ATCC), Rockville,Md., or other sources.

[0180] Influenza virus has a lipid bilayer envelope. The virions acquirethis membrane as they bud from the plasma membrane of an infected hostcell. Enveloped viruses, in general, utilize membrane fusion tointroduce their genome into the cytoplasm of new host cells duringsubsequent rounds of infection (see, e.g., White, Ann. Rev. Physiol.,52:675-697 (1990)). This fusion reaction may either occur at the levelof the host cell plasma membrane, or within acidic endosomes afteruptake of intact virions through receptor-mediated endocytosis. Duringendocytic cellular infection, the target membrane for fusion of theviral envelope is the limiting membrane of the endosomal cellcompartment.

[0181] Influenza membrane fusion capacity is activated only under mildlyacidic conditions. Low-pH-dependent viruses, such as influenza virus,must utilize the endocytic route of cellular infection for exposure tothe necessary acidic conditions, which they encounter in the lumen ofthe endosomes (Mellman, et al., Ann. Rev. Biochem., 55:663-700 (1986)).Fusion at the plasma membrane is precluded by the strict pH dependenceof their fusion activity. Infection of cells by low-pH-dependent virusescan be blocked by inhibitors of vacuolar acidification, such aschloroquine or NH₄Cl. In cultured cell systems influenza virus can beinduced to fuse with the cell plasma membrane by a transient lowering ofthe pH in the extracellular medium.

[0182] The influenza virus membrane contains two major integral spikeglycoproteins, hemagglutinin (HA) and neuraminidase (NA). The infectiousentry of the virions into the host cell is mediated by hemagglutinin.First, HA binds to sialic-acid-containing receptors on the cell surface.Second, following the internalization of the virus particles into theendosomal cell compartment (Stegmann, et al., Biochim. Biophys. Acta,904:165-170 (1987)), the HA also triggers the fusion reaction with theendosomal membrane.

[0183] The HA spike, protruding some 13.5 nm from the viral surface, isa homotrimeric molecule. Each monomer consists of two disulfide-linkedsubunits: HA1 (47 kD) and HA2 (28 kD), which are generated from a singlepolypeptide chain, HA0 (75 kD), by posttranslational cleavage by ahost-cell protease. The globular HA1 domains contain the sialic-acidbinding pockets. The N-terminus of HA2, generated by thepost-translational cleavage of HA0, appears crucial for the expressionof fusion activity of HA: Uncleaved HA0 is not fusion-active, whilesite-specific mutations within this region of the molecule severelyaffect the fusion activity of HA (Gething, et al., J. Cell Biol.,102:11-23 (1986)). The N-terminus of HA2, the so-called “fusionpeptide”, is a conserved stretch of some 20 amino acid residues that aremostly hydrophobic in nature (White, supra). At neutral pH the fusionpeptides are buried within the stem of the HA trimer about 3.5 nm fromthe viral surface. However, at low pH an irreversible conformationalchange in the HA results in their exposure (White and Wilson, J. CellBiol., 105:2887-2896(1987)).

[0184] Influenza virus envelopes, including the hemagglutinin, can besolubilized by treatment of virus particles with a detergent. Nonionicdetergents having a relatively low critical micellar concentration (CMC)are generally used to solubilize the envelope membranes.Octaethyleneglycol mono(n-dodecyl)ether (C₁₂E₈) and Triton X-100 may beused for solubilization, although other nonionic detergents may also beemployed.

[0185] One potential disadvantage of using low-CMC detergents forsolubilization and reconstitution of viral envelopes is that they cannot be easily removed from the system by, e.g., dialysis. Detergentswith a relatively high CMC such as N-octyl-β-D-glucopyranoside (octylglucoside; CMC of about 20 nM), may be used to solubilize influenzavirus envelopes. However, fusogenic virosomes are not readily preparedby subsequent removal of the octyl glucoside detergent. During dialysis,the hemagglutinin appears to concentrate primarily in lipid-pooraggregates with a very limited aqueous space, while the viral lipid isrecovered in protein-poor vesicles. Although these vesicles exhibit someHA-mediated membrane fusion activity, only a small fraction of the HA isrecovered in these vesicles (Stegmann, et al., supra).

[0186] To obtain virus for solubilization, influenza virus is grown tohigh titers on cultured cells (e.g., Madin-Darby Kidney cells, or MDCK)or in the allantoic cavity of 10-day-old embryonated chicken eggs. Topurify the virus from the allantoic fluid the harvested allantoic fluidis centrifuged (e.g., at 1000 g for 15 min in the cold) to removedebris, after which the virus is sedimented from the supernatant (e.g.,at 75,000 g for 90 min at 4°). The virus pellet is resuspended in buffersuch as “HNE” (150 mM NaCl, 0.1 mM EDTA, and 5 mM HEPES, adjusted to pH7.4) and subjected to sucrose gradient centrifugation (e.g., 10-60%,w/v, linear sucrose gradient in HNE at 100,000 g for 16 hr at 4°). Thevirus equilibrates as a single band at approximately 45% (w/v) sucrose.The band is collected, then frozen in small aliquots at −80°. Virus canalso be purified by a one-step affinity column chromatography, which isparticularly useful with virus which has been obtained from cellculture. The protein content of virus preparations can be determinedaccording to Peterson, Anal. Biochem., 83:346 (1977), incorporatedherein by reference, and the phospholipid content, after quantitativeextraction of the lipids from a known amount of virus, determinedaccording to Böttcher et al., Anal. Chim. Acta, 24:203 (1961),incorporated herein by reference.

[0187] For solubilization of viral envelopes a detergent such as, e.g.,C₁₂E₈ (Nikko Chemicals, Tokyo, Japan; Fluka, Buchs, Switzerland; orCalbiochem, San Diego, Calif.) is dissolved in HNE at a concentration ofabout 100 mM. BioBeads SM2 (Bio-Rad, Richmond, Calif.) or the like arewashed with methanol and subsequently with water, according to Holloway,Anal. Biochem., 55:304 (1973), incorporated herein by reference, andstored under water. Just before use the beads are drained on filterpaper and weighed. Sucrose solutions for gradient centrifugation aremade in HNE on a weight per volume basis.

[0188] A representative method for producing the virosomes of theinvention is now described, although it will be understood that theprocedure can be subjected to modifications in various aspects withoutaffecting the outcome. As described more fully below in the experimentalsection, influenza virus (the equivalent of about 1.5 μmol membranephospholipid) is diluted in HNE and sedimented (e.g., for 30 min at50,000 g in a Beckman Ti50 rotor) at 4°. HNE buffer containing detergentis added to the pellet (e.g., 0.7 ml of 100 mM C₁₂E₈) and the pelletresuspended and solubilization allowed to occur for another 15 min onice. Subsequently, the viral nucleocapsid is removed by centrifugation(e.g., for 30 min at 85,000 g at 4°) and a small sample of thesupernatant can be taken at this stage for protein and phospholipidanalysis. Of the initial viral protein and phospholipid, 35%(representing almost all of the membrane protein) and over 90%,respectively, may be recovered in the supernatant. The supernatant(e.g., 0.63 ml) is transferred to a 1.5-ml Eppendorf vial containingpre-washed BioBeads SM2 (e.g., 180 mg, wet weight) and the supernatantgently mixed with the beads. An additional amount of BioBeads (e.g., 90mg wet) is added and mixing continued. The formation of vesicularstructures is indicated when the suspension becomes turbid. Analternative procedure for removing the detergent from small volumes isaccording to Lundberg, et al., Biochim. Biophys. Acta, 1149:305 (1993),which is incorporated herein by reference. BioBeads are packed into aminicolumn and the preparation run through the column. A centrifugationprocedure or applying negative pressure can be used to force thepreparation through the column. The column procedure provides moreflexibility in terms of the ratio of the amount of BioBeads used and thevolume of the preparation. The virosome suspension is then centrifugedon a discontinuous sucrose gradient (e.g., 10-40% (w/v) for 90 min. at130,000 g at 4°), and the virosomes appear as a thin opalescent band andare collected from the interface between the two sucrose-containinglayers.

[0189] Other lipids can also be added to the virosome membranes duringpreparation. Fusion activity of the virosomes is optimally maintainedwhen lipids similar to those of viral origin or lipid mixtures whichclosely resemble the lipid composition of the viral envelope are added.These lipids comprise cholesterol and phospholipids such asphosphatidylcholine (PC), sphingomyelin (SPM), phosphatidylethanolamine(PE), and phosphatidylserine (PS). However, other phospholipids may alsobe added. These include, but are not limited to, phosphatidylglycerol(PG), phosphatidic acid (PA), cardiolipin (CL), and phosphatidylinositol(PI), with varying fatty acyl compositions and of natural and/or(semi)synthetic origin, and dicetyl phosphate. Ceramide and variousglycolipids, such as cerebrosides or gangliosides, may also be added.Cationic lipids may also be added, e.g., for concentrating nucleic acidsin the virosomes and/or for facilitating virosome-mediated delivery ofnucleic acids to cells. These include DOTMA, DOTAP(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride), DODAC(N,N-dioleyl-N,N, dimethylammonium chloride), DDAB and stearylamine orother aliphatic amines and the like. DODAC is a preferred cationic lipidfor complexing nucleic acids to the virosome and the ensuing delivery ofnucleic acids to cells, and is described in copending application U.S.Ser. No. 08/316,399, incorporated herein by reference. Particularlypreferred concentrations of DODAC range from 25-45% (mol % of totalphospholipids in the virus), more preferably 30-40%, and most preferablyabout 30% for the delivery of a nucleic acid such as DNA or antisenseRNA to a cell. Additional lipids which may be suitable for use in thevirosomes of the present invention are well known to persons of skill inthe art. Nucleic acids such as oligonucleotides and DNA can also beencapsulated in virosomes after condensation with polylysine to formparticles that are then enclosed within a virosome for delivery to acell rather than being complexed to it, thereby minimizing or avoiding,if desired, the use of a cationic lipid. Furthermore, encapsulated DNAis protected from DNase degradation.

[0190] Typically, in a virosome preparation procedure involvingadditional lipids, the additional lipids are dried from a mixed solutionin chloroform/methanol to a film at the bottom of a tube by evaporationof the solvent and subsequent exposure to vacuum for 1 h. Then thesupernatant fraction obtained after solubilization of the viral envelopein detergent (e.g., C₁₂E₈) and sedimentation of the nucleocapsid byultracentrifugation is added to the film. The quantities of additionallipid and supernatant are chosen such that the desired ratio of viral toadditional lipid is obtained. The detergent is then removed viatreatment with BioBeads or the like as described above.

[0191] Generally, the virosomes should resemble a viral envelope instructure and composition as closely as possible. The virosomepreparation should generally consist of a relatively uniform populationof vesicles in terms of size and protein-to-lipid ratio. Residualdetergent should be minimal and not interfere with virosome function.The virosomes should mimic the biological activity of the native viralenvelope. Generally, the virosomes should exhibit pH-dependent membranefusion activity.

[0192] Virosomes can also be prepared with viral fusion proteins havingdifferent pH sensitivities, derived from, e.g., different influenzavirus strains. The different pH sensitivities of the virosome can betaken advantage of to prepare virosome-liposome hybrids that encapsulateand deliver large therapeutic molecules such as DNA or proteins that maybe difficult to encapsulate directly and with high efficiency invirosomes prepared according to the above protocol. A liposome is firstprepared which encapsulates the therapeutic agent with high efficiency.The liposome is then fused with the virosome at the pH of the viralmembrane fusion protein having the higher pH threshold for fusion. Thisresults in a virosome-liposome hybrid containing the encapsulatedtherapeutic agent. The virosome-liposome hybrid is then used to deliverthe encapsulated therapeutic agent to the cytosol of cells by fusiontriggered at the pH of the viral fusion protein with the lower pHthreshold for fusion.

[0193] The incorporation of hemagglutinin in reconstituted vesicles isreadily assessed by equilibrium density-gradient analysis. The virosomepreparation, collected from the discontinuous sucrose gradient, isdiluted with HNE and applied to a linear sucrose gradient in HNE (e.g.,10-60% (w/v)) and the gradient centrifuged (e.g., at 170,000 g for 30 hrat 4°), after which fractions are collected and analyzed for protein andphospholipid content. The virosomes appear as a single band, containingboth protein and phospholipids. The density of the virosomes will differdepending on the presence of additional lipids. In general, the densitywill decrease when the ratio of additional lipids to viral lipidsincreases.

[0194] Analysis of influenza virosomes by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) may be performedto confirm that the virosomes contain the hemagglutinin protein. Theviral nucleoprotein NP, the matrix protein M1 the minor integralmembrane protein of influenza virus, M2, are generally not detectable insuch analysis. The virosomes have a protein-to-(phospho)lipid ratio thatis similar to the ratio in the solubilization mixture aftersedimentation of the nucleocapsid, but which will change when additionallipid is added to the virosome preparation.

[0195] Recovery of viral membrane protein and phospholipid in thevirosome preparation ranges from 30 to 50% relative to the initiallysolubilized material. Residual detergent in virosomes prepared accordingto the above protocol is typically about 7.5 mol % relative to the totalvirosomal lipid. This level of detergent generally does notsignificantly affect the fusogenic activity of the virosomes, butresidual detergent may have an effect on the capacity of the virosomesto retain low-molecular-weight encapsulated compounds.

[0196] Negative-stain electron microscopy (EM) is the most widelyapplied and accessible technique for assessing the structure and size ofvirosomes. The staining solution preferably has a neutral pH, so as toavoid acid-induced conformational changes of the hemagglutinin protein.Briefly, a droplet of the virosome suspension, after dialysis againstisotonic ammonium acetate buffered to neutral pH with 5 mM HEPES, isapplied to a grid with a carbon-coated Formvar film, afterglow-discharge of the grid. The specimen is placed upside down for 1 minon a droplet of 2% phosphotungstic acid (PTA) at neutral pH (or, e.g.,1% sodium silicotungstate of neutral pH), drained and dried in air.

[0197] Fusion of virosomes with biological or artificial targetmembranes can be followed with a fluorescent resonance energy transferassay (RET). In a convenient assay,N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (N-NBD-PE)is used as a donor probe and N-(lissamine rhodamine B sulfonyl)PE(N-Rh-PE) as the acceptor. A variant of this assay, utilizing the samedonor N-NBD-PE but a different acceptor,cholesterol-anthracene-9-carboxylate (CAC), may also be used. Uponfusion of a membrane, labeled with the N-NBD-PE/N-Rh-PE pair, the twofluorophores dilute into the target membrane, resulting in a decrease oftheir overall surface density and a concomitant decrease of the RETefficiency. This decrease can be followed as an increase of the donor(N-NBD-PE) fluorescence. This assay can be used to assess pH-dependentfusion of influenza virosomes with a membrane, including, e.g.,erythrocyte ghosts and BHK cells.

[0198] Another in vitro means to assess fusion of virosomes is anexcimer assay using pyrene-labeled lipids. Pyrene fluorophores may formexcited dimers (excimers) between a probe molecule in the excited stateand a probe molecule in the ground state. The fluorescence emission ofthe excimer is shifted to higher wavelengths by about 100 nm relative tothe emission of the monomer. Excimer formation is dependent on thedistance between the probe molecules. Thus, coupled to one of the acylchains of a phospholipid molecule, such as phosphatidylcholine (PC), thepyrene probe provides a sensitive measure of the surface density of thelabeled molecules in a lipid bilayer membrane. On fusion of apyrene-PC-labeled membrane with an unlabeled membrane, the pyrene-PCsurface density decrease can be monitored as a reduction of the excimerfluorescence.

[0199] The RET probes N-NBD-PE/N-Rh-PE or the Pyrene-PC probe (MolecularProbes, Eugene, Oreg.) are incorporated in the virosomal membrane asfollows. The supernatant obtained after solubilization of the viralmembrane and sedimentation of the nucleocapsid (see above) is added to adry film of the probe (10 mol % with respect to the viral lipid). Themixture is lightly shaken to allow mixing of the probe with the virallipids, and detergent is removed as described above.

[0200] Fusion of the labeled virosomes can be conveniently measuredusing resealed human erythrocyte ghosts as a model biological targetmembrane system. Alternatively, fusion activity toward liposomes can beassessed, in which case it is important to avoid liposomes consistingprimarily of negatively charged phospholipids, such as cardiolipin, asthese appear to support a fusion reaction with influenza virus orvirosomes, whose characteristics deviate from those of fusion withbiological membranes. Fusion with liposomes consisting of a 2:1 mixtureof PC and PE (Avanti Polar Lipids, Alabaster, Ala.), and containing 5mol % of the ganglioside G_(Dta) or total bovine brain gangliosides(Sigma Chemical Co., St. Louis, Mo.) serving as sialic acid-containingreceptors for the virus/virosomes, provides a convenient assay. Fusionmay also be monitored in an on-line fashion using cultured cells astargets. Either endocytic uptake of the virosomes at neutral pH andsubsequent fusion from within endosomes or direct fusion with the cellplasma membrane induced by a transient lowering of the extracellular pHmay be used.

[0201] An alternative to direct assessment of the fusion activity ofinfluenza virosomes is determining their hemolytic activity. The fusionactivity of influenza virosomes, produced according to the proceduredescribed above, typically corresponds closely to hemolytic activity,exhibiting a pH dependence identical to that of fusion. Hemolyticactivity of influenza virosomes may be determined by, for example,adding the virosomes (the equivalent of 1 nmol of phospholipid, in avolume of 25 μl) to 4×10⁷ washed human erythrocytes in 975 μl fusionbuffer (135 mM NaCl, 15 mM sodium citrate, 10 mM MES, 5 mM HEPES), setto various pH values. After incubation at 37° for 30 min, the mixture iscentrifuged for 3 min at 1350 g. Lysis of erythrocytes is quantified bythe measurement of absorbance of the hemoglobin in the supernatant at541 nm. Maximal hemolysis is determined after lysis of the erythrocytesin distilled water.

[0202] Additional components may be added to the virosomes to target thevirosomes to specific cell types. For example, the virosomes can beconjugated to monoclonal antibodies that bind to epitopes present onlyon specific cell types. For example, monoclonal antibodies may bindspecifically to cancer-related antigens providing a means for targetingthe virosomes following systemic administration. Alternatively, ligandsthat bind surface receptors of the target cell types may also be boundto the virosomes. Other means for targeting liposomes may also beemployed in the present invention.

[0203] The fusogenic virosomes are employed to carry therapeuticcompounds for introduction into cells. As used herein, “therapeuticcompound” is meant to indicate a synthetic compound suitable fortherapeutic use. “Therapeutic compound” is meant to include, e.g.,nucleic acids (antisense, DNA), proteins, peptides, oncolytics,anti-infectives, anxiolytics, psychotropics, ionotropes, toxins such asgelonin and inhibitors of eucaryotic protein synthesis, and the like.“Synthetic compounds” are compounds that are not naturally occurring orcompounds that are isolated from the environment in which they naturallyoccur.

[0204] The therapeutic compound may be carried in the aqueous interiorof the virosome or in the lipid membrane of the virosome. A variety oftherapeutic compounds may be carried in the virosomes of the presentinvention. The virosomes provide a means for facilitated entry of thetherapeutic compounds into the cells:

[0205] Particularly useful is encapsulation of therapeutic compoundsthat are active within the cytoplasm of host cells. Such compoundsinclude, e.g., DNA encoding proteins or peptides operably linked to apromoter active in the host cell, RNA encoding a protein or peptide,nucleic acids such as antisense oligonucleotides (as described in, e.g.,WO 93/09813 and WO 93/01286, both incorporated herein by reference) andribozymes (e.g., U.S. Pat. Nos. 4,987,071, 5,254,678, and WO 94/26877,each incorporated herein by reference), oncolytic agents,anti-inflammatory agents, cardiovascular agents, anti-infective agents,psychotropic agents, and the like. The therapeutic compounds aredelivered into the host cell cytoplasm upon fusion of the virosome withthe endosome or plasma membrane.

[0206] The therapeutic compounds will generally be foreign to the host.By “foreign,” it is meant a compound that is not naturally present inthe host. Alternatively, the therapeutic compound may not be foreign tothe host. The compound may naturally occur within the host. For example,nucleic acids encoding a naturally occurring protein may be introducedinto host cells to increase expression of the protein in the cells. Thenucleic acid can be either DNA or RNA. For expression, the nucleic acidwill typically comprise at least the following operably linked elements:a transcriptional promoter, a gene encoding the desired therapeuticprotein, and a transcriptional terminator.

[0207] Therapeutic compounds may be incorporated into the virosome atthe time of virosome preparation. Typically, the therapeutic compound isadded to the lipid/hemagglutinin-containing solution following removalof the nucleocapsid. Alternatively, the therapeutic compound isencapsulated in a virosome-liposome hybrid by initial encapsulation ofthe compound in a liposome, followed by fusion of the liposome with avirosome containing two hemagglutinins with different pH thresholds forfusion, as outlined above.

[0208] For administration to host cells the virosomes are carried in apharmaceutically acceptable carrier. Many pharmaceutically acceptablecarriers may be employed in the compositions of the present invention.Generally, normal buffered saline (135-150 mM NaCl) will be employed asthe pharmaceutically acceptable carrier, but other suitable carrierswill suffice. These compositions may be sterilized by conventionalliposomal sterilization techniques, such as filtration. The compositionsmay contain pharmaceutically acceptable auxiliary substances as requiredto approximate physiological conditions, such as pH adjusting andbuffering agents, tonicity adjusting agents and the like, for example,sodium acetate, sodium lactate, sodium chloride, potassium chloride,calcium chloride, etc.

[0209] The concentration of virosomes in the carrier may vary.Generally, the concentration will be about 20-200 mg/ml, usually about50-150 mg/ml, and most usually about 75-125 mg/ml, e.g., about 100mg/ml. Persons of skill may vary these concentrations to optimizetreatment with different virosome components or for particular patients.For example, the concentration may be increased to lower the fluid loadassociated with treatment; This may be particularly desirable inpatients having atherosclerosis-associated congestive heart failure orsevere hypertension.

[0210] The present invention also provides methods for introducingtherapeutic compounds into cells of a host. The methods generallycomprise contacting the cells of the host with a virosome containing thetherapeutic compound, wherein the virosome has a membrane and an aqueousinterior, and a viral membrane fusion protein, e.g., influenzahemagglutinin, is contained in the membrane. The host may be a varietyof animals, including humans, non-human primates, avian species, equinespecies, bovine species, swine, lagomorpha, rodents, and the like.

[0211] The cells may be contacted by in vivo administration of thevirosomes or ex vivo contacting of the virosomes to the cells. In vivocontact is obtained by administration of the virosomes to host. Thevirosomes may be administered in many ways. These include parenteralroutes of administration, such as intravenous, intramuscular,subcutaneous, and intraarterial. Generally, the virosomes will beadministered intravenously or via inhalation. Often, the virosomes willbe administered into a large central vein, such as the superior venacava or inferior vena cava, to allow highly concentrated solutions to beadministered into large volume and flow vessels. The virosomes may beadministered intraarterially following vascular procedures to deliver ahigh concentration directly to an affected vessel. The virosomes mayalso be administered topically. In some instances, the virosomes may beadministered orally or transdermally. The virosomes may also beincorporated into implantable devices for long term release followingplacement.

[0212] As described above, the virosomes are typically administeredintravenously or via inhalation in the methods of the present invention.Often multiple treatments will be given to the patient. The dosageschedule of the treatments will be determined by the disease and thepatient's condition. Standard treatments with therapeutic compounds thatare well known in the art may serve as a guide to treatment withvirosomes containing the therapeutic compounds. The duration andschedule of treatments may be varied by methods well known to those ofskill.

[0213] The dose of virosomes of the present invention may vary dependingon the clinical condition and size of the animal or patient receivingtreatment. The standard dose of the therapeutic compound when notencapsulated may serve as a guide to the dose of thevirosome-encapsulated compound. The dose will typically be constant overthe course of treatment, although the dose may vary in some instances.Standard physiological parameters may be assessed during treatment thatmay alter the dose of the virosomes.

[0214] VII. EXAMPLES

[0215] A. Examples Relating to Fusogenic Liposomes Containing BilayerStabilizing Components

[0216] 1. MATERIALS AND GENERAL METHODS

[0217] a. Materials

[0218] All phospholipids including fluorescent probes and PEG-PEconjugates were purchased from Avanti Polar Lipids, Birmingham, Ala.,USA. 1-O-methyl-(poly(ethoxy)-O-succinyl-O-(egg)ceramide which was agift from Dr L. Choi of Inex Pharmaceuticals Corp., Vancouver, BC,Canada. Di-[1-¹⁴C]-palmitoylphosphatidyl-choline was purchased fromDuPont, Mississuaga, Ont., Canada. [³H]-DSPE-PEG₂₀₀₀ was synthesized asdescribed previously (Parr, et al., Biochim. Biophys. Acta, 1195: 21-30(1994)). Other reagents were purchased from Sigma Chemical Co., StLouis, Mo., USA.

[0219] b. Preparation of Multilamellar Vesicles and Large UnilamellarVesicles

[0220] Lipid components were mixed in 1-2 ml of benzene:methanol (95:5,v/v) and then lyophilized for a minimum of S hours at a pressure of <60millitorr using a Virtis lyophilizer equipped with a liquid N₂ trap.Multilamellar vesicles (MLVs) were prepared by hydrating the dry lipidmixtures in 150 mM NaCl, buffered with 10 mM Hepes-NaOH, pH 7.4(Hepes-buffered saline, HBS). Mixtures were vortexed to assisthydration. To produce large unilamellar vesicles (LUVs), MLVs were firstfrozen in liquid nitrogen and then thawed at 30° C. five times. LUVswere produced by extrusion of the frozen and thawed MLVs ten timesthrough 2 stacked polycarbonate filters of 100 nm pore size at 30° C.and pressures of 200-500 psi (Hope, et al., Biochim. Biophys. Acta,812:55-65 (1985)).

[0221] c. ³¹P-NMR spectroscopy

[0222]³¹P-NMR spectra were obtained using a temperature controlledBruker MSL200 spectrometer operating at 81 MHz. Free induction decayswere accumulated for 2000 transients using a 4 μs, 90° pulse, 1 sec.interpulse delay, 20 KHz sweep width and Waltz decoupling. A 50 Hz linebroadening was applied to the data prior to Fourier transformation.Samples were allowed to equilibrate at the indicated temperature for 30minutes prior to data accumulation. Lipid concentrations of 30-70 mMwere used.

[0223] d. Freeze-Fracture Electron Microscopy

[0224] MLVs were prepared by hydrating a mixture ofDOPE:cholesterol:DOPE-PEG₂₀₀₀ (1:1:0:1) with HBS. A portion of themixture was extruded as described above to produce LUVs. Glycerol wasadded to both MLVs and LUVs to a final concentration of 25% and sampleswere rapidly frozen in liquid freon. The samples were fractured at −110°C. and <10⁻⁶ torr in a Balzers BAF400 unit. Replicas were prepared byshadowing at 45° with a 2 nm layer of platinum and coating at 90° with a20 nm layer of carbon. The replicas were cleaned by soaking inhypochlorite solution for up to 48 hrs and were visualized in a JeolJEM-1200 EX electron microscope.

[0225] e. Gel Filtration of LUVs and Micelles

[0226] LUVs composed of DOPE:cholesterol:DSPE-PEG₂₀₀₀ (1:1:0:1) withtrace amounts of ¹⁴C-DPPC and ³H-DSPE-PEG₂₀₀₀ were chromatographed at aflow rate of approximately 0.5 ml/min on a column of Sepharose CL-4B waspretreated with 10 mg of eggPC, which had been suspended in HBS by bathsonication, to eliminate non-specific adsorption of lipid to the column.Micelles were prepared by hydrating DSPE-PEG₂₀₀₀ containing a traceamount of ³H-DSPE-PEG₂₀₀₀ with HBS and chromatographed as described forLUVs.

[0227] f. Lipid Mixing Assays

[0228] Lipid mixtures were prepared as described for NMR measurements.The resultant multilamellar vesicles (MLV) were frozen in liquidnitrogen and then thawed at 30° C. five times. Large -unilamellarvesicles (LUV) were produced by extrusion of the frozen and thawed MLVten times through 2 stacked polycarbonate filters of 100 mn pore size at30° C. and pressures of 200-500 psi (Hope, et al., Biochim. Biophys.Acta, 812:55-65 (1985)).

[0229] Lipid mixing was measured by a modification of the fluorescenceresonance energy transfer (FRET) assay of Struck, et al. (Biochemistry,20:4093-4099 (1981)). LUVs were prepared containing the fluorescentlipids,N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-dioleoylphosphatidylethanolamine(NBD-PE) and N-(lissamine rhodamine Bsulfonyl)-dipalmitoylphosphatidylethanolamine (Rh-PE) at 0.5 mol %. LUVs(50-60 μM) and a three-fold excess of unlabelled target vesicles weremixed in the fluorimeter at 37° C. for short term assays (≦1 hour), orin sealed cuvettes in a dark water bath at 37° C. for longer assays. Formeasurements of fusion after PEG-lipid transfer, an excess of liposomesprepared from 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) was addedas a sink for the PEG-lipid. Fluorescence emission intensity wasmeasured at 517 nm with excitation at 465 nm both before and after theaddition of Triton X-100 (final concentration of 0.5% or 1% when POPCsink was used). Data is presented as either uncorrected fluorescenceintensity for short term assays (≦1 hour) or as percentage fusion. Lightscattering controls were performed by replacing LUVs labelled with 0.5mol % probes with unlabelled vesicles. Maximum fusion was determinedusing mock fused vesicles containing 0.125 mol % of each fluorescentprobe. The percentage fusion was calculated as:${\% \quad {Fusion}} = {\frac{\frac{\left( {F_{(t)} - L_{(t)}} \right)}{\left( {F_{T} - L_{T}} \right)} - \frac{\left( {F_{o} - L_{o}} \right)}{\left( {F_{T} - L_{T}} \right)}}{\frac{\left( {M_{(t)} - L_{(t)}} \right)}{\left( {M_{T} - L_{T}} \right)} - \frac{\left( {F_{o} - L_{o}} \right)}{\left( {F_{T} - L_{T}} \right)}} \times 100}$

[0230] where F_((t))=fluorescence intensity at time t;F_(o)=fluorescence intensity at zero time; F_(T)=fluorescence intensityin the presence of Triton X-100. M and L represent the same measurementsfor the mock fused control and the light scattering controlrespectively. Changes in fluorescence of the mock fused controlindicated that exchange of the fluorescent probes over 24 hoursaccounted for 10% of the fluorescence change observed, but wasnegligible over the first hour.

[0231] g. Fusion of Liposomes with Red Blood Cells

[0232] LUVs composed of DOPE:cholesterol:DODAC (40:45:15) orDOPE:cholesterol:DODAC:PEG-ceramide (35:45:15:) were prepared bystandard extrusion techniques. LUVs also contained 1 mol % rhodamine-PE.LUVs (200 μM) were incubated at 37° C. with 50 μl packed RBCs in a finalvolume of 1 ml. For assays of fusion after PEG-lipid exchange, a sink of2 mM POPC:cholesterol (55:45) was included. In some assays, thefusogenic liposomes were pre-incubated with the sink before being mixedwith the RBCs (See, figure legends for FIGS. 22-24). Aliquots of themixtures were transferred to glass microscope slides, covered with coverslips and examined by phase contrast and fluorescent microscopy. Fusionwas assessed as fluorescent labeling of the RBC plasma membranes. ForFIGS. 22-24, fluorescent liposomes were incubated with POPC:cholesterolliposomes and/or RBCs as described in section “L,” infra. Panels a,c ande of FIGS. 22-24 are views under phase contrast, whereas panels b,d andf of FIGS. 22-24 are the same fields viewed under fluorescent light.

[0233] h. Other Procedures

[0234] Phospholipid concentrations were determined by assaying forphosphate using the method of Fiske and Subbarow (J. Biol. Chem.,66:375-400 (1925)). Liposome size distributions were determined byquasi-elastic light scattering (QELS) using a Nicomp model 370 particlesizer.

[0235] 2. EXPERIMENTAL FINDINGS

[0236] a. Influence of BSC on the Polymorphic Phase Properties of anEquimolar Mixture of DOPE and Cholesterol

[0237]³¹P-NMR was used to examine the effect of bilayer stabilizingcomponent (BSC), in this instance poly-(ethyleneglycol)₂₀₀₀ conjugatedto DOPE (i.e., DOPE-PEG₂₀₀₀), on the phase preference of an equimolarmixture of DOPE and cholesterol (FIG. 1). In the absence of BSC, themixture adopted an inverse hexagonal phase (H_(II)) at 20° C. asdetermined from the characteristic ³¹P-NMR lineshape with a low fieldpeak and high field shoulder (Cullis and deKruijff, Biochim. Biophys.Acta 559:399-420 (1979)). As the amount of BSC in the mixture wasincreased, the peak corresponding to H_(II) phase phospholipiddisappeared and a high field peak with a low field shoulder,characteristic of bilayer phase phospholipid (Cullis and deKruijff,supra, 1979) appeared. When DOPE-PEG₂₀₀₀ was present at 20 mol % ofphospholipid, the mixture was almost exclusively bilayer with noevidence of H_(II) phase lipid.

[0238] In addition to the peaks corresponding to H_(II) phase andbilayer phase, a third peak indicative of isotropic motional averagingwas observed in the presence of BSC (FIG. 1). The size of the isotropicsignal varied with the amount of BSC present and, as shown in subsequentFigures, the nature of the BSC species. The signal was largest atconcentrations of BSC that allowed H_(II) and bilayer phases to co-existand diminished when either H_(II) or bilayer phase predominated. Such asignal may be produced by a number of phospholipid phases which allowisotropic motional averaging on the NMR timescale, including micellar,small vesicular, cubic and rhombic phase phospholipids.

[0239] b. The Influence of BSC on the Thermotropic Properties of anEquimolar Mixture of DOPE and Cholesterol

[0240]FIG. 2 illustrates the effect of temperature on the phaseproperties of mixtures of DOPE, cholesterol and BSC. When DOPE-PEG₂₀₀₀was present at 9 mol %, there was a large isotropic signal whichdominated the spectrum at all temperatures. The predominant,non-isotropic phase at 0° C. was bilayer. However, as the temperaturewas increased the high field peak diminished and a shouldercorresponding to the low field peak of the H_(II) phase appeared. Theapparent bilayer to hexagonal phase transition occurred at 40-50° C.,but was almost obscured by the large isotropic signal. DOPE on its ownexhibits a sharp transition over an interval of approximately 10° C.(see, FIG. 1 in Tilcock, et al., Biochemistry, 21:4596-4601 (1982)). Thetransition in mixtures of DOPE, cholesterol and BSC was slow incomparison with both phases present over a temperature range of almost40° C. (See also, FIG. 3).

[0241] The mixture was stabilized in the bilayer conformation over thesame temperature range when the BSC content was increased to 20 mol %(FIG. 2). There was no evidence of phospholipid in the H_(II) phase. Inaddition, the isotropic signal was markedly reduced at the higher BSCconcentration at all temperatures studied. The amount of lipidexperiencing isotropic motional averaging increased as the temperatureincreased for both concentrations of BSC.

[0242] c. The Effect of Head Group Size on the Bilayer StabilizingProperties of BSCs

[0243] The influence of head group size on the bilayer stabilizingproperties of BSCs is illustrated in FIG. 3. DOPE-PEG₂₀₀₀ at 5 mol % hadlimited bilayer stabilizing ability. A broad bilayer to H_(II)transition was centered at approximately 10° C., but a large proportionof the lipid adopted non-bilayer phases at all temperatures examined.Increasing the size of the headgroup by using poly-(ethyleneglycol)₅₀₀₀conjugated to DOPE (DOPE-PEG₅₀₀₀) in place of DOPE-PEG₂₀₀₀, at the samemolar fraction, caused a marked increase in bilayer stability. Thebilayer to H_(II) transition temperature increased to approximately 30°C. and the isotropic signal was barely discernible. The broadening ofthe bilayer to H_(II) transition noted above is particularly evidenthere with H_(II) phase lipid present at 0° C. and bilayer phase lipidpresent at 40° C.

[0244] d. The Influence of Acyl Chain Composition on the BilayerStabilizing Properties of BSCs

[0245] The bilayer stabilizing ability of three BSCs differing only inacyl chain composition is shown in FIG. 4. PEG₂₀₀₀ conjugated todimyristoylphosphatidyl-ethanolamine (DMPE-PEG₂₀₀₀),dipalmitoylphosphatidylethanolamine (DPPE-PEG₂₀₀₀) ordistearoylphosphatidylethanolamine (DSPE-PEG₂₀₀₀) showed a similarability to stabilize an equimolar mixture of DOPE and cholesterol. Thebilayer to H_(II) phase transition was raised to approximately 40-50° C.The results are similar to those presented in FIG. 2 which were obtainedusing a BSC with the same headgroup, but unsaturated acyl groups(DOPE-PEG₂₀₀₀) at the same concentration. The size of the isotropicsignal varied somewhat with the different BSCs, being smallest withDSPE-PEG₂₀₀₀ and largest with DOPE-PEG₂₀₀₀ (cf., FIG. 2 and FIG. 4).

[0246] e. The Use of PEG-Ceramides as Bilayer Stabilizing Components

[0247] The spectra set forth in FIGS. 1-4 were all obtained using PEGconjugated to phosphatidylethanolamine through a carbamate linkage. Inaddition, however, the use of ceramide as an alternative anchor for thehydrophilic polymer was examined. PEG₂₀₀₀ was conjugated via a succinatelinker to egg ceramide. FIG. 5 shows the ³¹P-NMR spectra obtained usingmixtures of DOPE:cholesterol:egg ceramide-PEG₂₀₀₀ (1:1:10.1 and1:1:0.25) over the temperature range of 0 to 60° C. At the lower molarratio of PEG-ceramide, both bilayer and H_(II) phase lipid are inevidence at most temperatures. However, at the higher PEG-ceramide molarratio, the spectra are exclusively bilayer up to 60° C. at which point alow field shoulder corresponding to H_(II) phase lipid is visible.Unlike the spectra obtained using PEG-PEs, there was almost no isotropicsignal when PEG-ceramide was used.

[0248] f. Freeze-Fracture Electron Microscopy

[0249] One of the interesting features of several of the NMR spectra wasthe narrow signal at 0 ppm, indicative of isotropic motional averaging.This signal can arise from a number of phospholipid phases such asmicellar, small vesicular, cubic and rhombic phase structures.Freeze-fracture electron microscopy was used to investigate this aspectfurther. FIG. 6 shows an electron micrograph of MLVs prepared byhydrating a mixture of DOPE:cholesterol:DOPE-PEG₂₀₀₀ (1:1:0.1) with HBSat room temperature. This lipid mixture corresponds to the NMR spectraset forth in FIG. 2A which exhibited evidence of bilayer, H_(II) andisotropic phases.

[0250] A number of different structures are visible in the micrograph.Much of the lipid is present as large spherical vesicles of 400 to 600nm in diameter. Many of the vesicles have indentations which appear tobe randomly distributed in some vesicles, but organized in straight orcurved lines in others. Cusp-like protrusions are also visible on theconcave surfaces of some vesicles. These features are commonly referredto as lipidic particles (Verkleij, A. J., Biochim. Biophys. Acta,779:43-92 (1984)) and may represent an intermediate structure formedduring fusion of bilayers. These large vesicles would be expected togive rise to a predominately bilayer ³¹P-NMR spectrum with a narrowisotropic signal due to the lipidic particles. Similar results have beenobserved with N-methylated PEs (Gagne, et al., Biochemistry,24:4400-4408 (1985)). A number of smaller vesicles of around 100 nmdiameter can also be seen. These vesicles may have been formedspontaneously on hydration, or may have been produced by vesiculizationof larger vesicles. These vesicles are sufficiently small for lipidlateral diffusion, or tumbling of the vesicles in suspension, to producemotional averaging on the NMR timescale (Burnell, et al., Biochim.Biophys. Acta, 603:63-69 (1980)), giving rise to an isotropic signal(see, FIG. 2A). In the center of FIG. 6 is a large aggregate showingevidence of several different structures. The right side of theaggregate is characterized by what appears to be closely packed lipidicparticles. The upper left hand side shows a distinct organization intothree-dimensional cubic arrays and the lower left hand region has theappearance of stacked tubes characteristic of lipid adopting the H_(II)phase (Hope, et al., J. Elect. Micros. Tech., 13:277-287 (1989)). Thisis consistent with the corresponding ³¹P-NMR spectrum.

[0251]FIG. 7 shows the appearance of the same mixture after extrusionthrough polycarbonate filters of 100 nm pore size to produce LUVS. Thelipid is predominately organized into vesicles of approximately 100 nmin diameter. Closer inspection reveals the presence of occasional largervesicles and some of tubular shape. Overall the fairly uniform sizedistribution is typical of that obtained when liposomes are produced byextrusion.

[0252] The presence of lipid micelles is not readily apparent fromfreeze fracture electron microscopy. Lipid in the micellar phase could,however, contribute to the isotropic signal observed in NMR spectra, andit has previously been shown that PEG-PE conjugates form micelles whenhydrated in isolation (Woodle and Lasic, Biochim. Biophys. Acta,113:171-199 (1992)). As such, the presence of micelles was tested bysubjecting a suspension of LUVs to molecular sieve chromatography onSepharose 4B. The liposomes were of the same composition used for thefreeze fracture studies above except that DSPE-PEG₂₀₀₀ was used in placeof DOPE-PEG₂₀₀₀, and they contained trace amounts of ¹⁴C-DPPC and³H-DSPE-PEG₂₀₀₀. The elution profile is shown in FIG. 8. A single peakcontaining both the phospholipid and PEG-PE conjugate markers was foundin the void volume. A control experiment also shown in FIG. 8demonstrated that micelles, which formed when PEG-PE was hydrated inisolation, were included into the column and would have been clearlyresolved if present in the liposomal preparation.

[0253] g. Effect of PE-PEG₂₀₀₀ On Fusion Of PE:PS LUVs

[0254] When unlabelled LUVs composed of DOPE: POPS (1:1) were added tofluorescently labelled LUVs there was a small jump in fluorescenceintensity due to increased light scattering but no fusion (FIG. 9, tracea). Upon addition of 5 mM Ca²⁺, there was a rapid increase influorescence consistent with lipid mixing as a result of membranefusion. Fusion was complete within a few seconds and was followed by aslow decrease in fluorescence. Inspection of the cuvette revealed thepresence of visible aggregates that settled despite stirring, resultingin the observed decrease in fluorescence. When PEG₂₀₀₀ conjugated todimyristoylphosphatidylethanolamine (DMPE-PEG₂₀₀₀) was included in bothvesicle populations, however, inhibition of fusion was observed. Asshown in FIG. 9 (traces b-d), inhibition was dependent on theconcentration of DMPE-PEG₂₀₀₀ in the vesicles with as little as 2 mol %being sufficient to eliminate Ca²⁺-induced fusion.

[0255] h. The Effect of PE-PEG Loss on Fusion

[0256] Recently, it has been demonstrated that phospholipids conjugatedto PEG of molecular weights 750-5,000 Da have enhanced rates ofspontaneous transfer between liposomes. The half-time for transfer ofthese conjugates can vary from minutes to hours and depends on both thesize of the PEG group and the nature of the acyl chains which anchor theconjugate in the bilayer. As such, fusion was examined under conditionswhere the PEG-lipid would be expected to transfer out of the liposomes.Ca²⁺ ions were added to PE:PS liposomes containing 2 mol % DMPE-PEG₂₀₀₀,followed by a twelve-fold excess (over labelled vesicles) of1-paimitoyl-2-oleoyl-phosphatidylcholine (POPC) liposomes as a sink forthe PEG-PE. As shown in FIG. 10 (curve a), while fusion was initiallyblocked by the presence of DMPE-PEG₂₀₀₀, the addition of POPC liposomes,which acted as a sink, lead to recovery of full fusogenic activityfollowing a short time lag. The small initial jump in fluorescenceintensity that occurred when POPC liposomes were added to PE:PSliposomes resulted from increased light scattering, not fusion. Controlexperiments demonstrated that no fusion occurred between the PE:PSliposomes and the POPC liposomes (data not shown), and no fusionoccurred in the absence of POPC liposomes (FIG. 10, curve b).

[0257] To confirm that recovery of fusogenic activity was dependent onremoval of the PEG-PE, the influence of initial PEG-lipid concentrationon the duration of the lag phase prior to fusion was examined (FIG. 11).Liposomes containing equimolar PE and PS were prepared with 2, 3, 5 or10 mol % DMPE-PEG₂₀₀₀. Fluorescently labelled and unlabelled vesicleswere mixed at a ratio of 1:3 and after the addition of 5 mM CaCl₂, a36-fold excess (over labelled vesicles) of POPC liposomes was added. Asexpected, there was an increase in the time delay prior to fusion withincreasing PEG-lipid concentration.

[0258] i. The Effect of Conjugate Acyl Chain Composition on FusogenicActivity

[0259] Since fusion is dependent on prior transfer of the PEG-PE out ofthe liposomes, it was thought that the rate at which fusogenic activitywas recovered would depend on the rate of transfer of the PEG-PE. Onefactor that affects the rate at which a phospholipid transfers from onemembrane to another is the length of its acyl chains. As such, theeffect of conjugate acyl chain composition on fusogenic activity wasinvestigated. In doing so, the recovery of fusogenic activity of PE:PSLUVs containing 2 mol % DMPE-PEG₂₀₀₀ was compared with PE:PS LUVscontaining 2 mol % DPPE-PEG₂₀₀₀ and 2 mol % DSPE-PEG₂₀₀₀ (FIG. 12A).Increasing the length of the acyl chains from 14 to 16 carbons caused adramatic increase in the lag period before fusion was initiated.Although the same level of fusion occurred using either DMPE-PEG₂₀₀₀ orDPPE-PEG₂₀₀₀, it was essentially complete in 40 minutes whenDMPE-PEG₂₀₀₀ was the stabilizer, but took 24 hours when DPPE-PEG₂₀₀₀ wasused. The implied decrease in rate of transfer (30-40 fold) isconsistent with previous measurements of the effect of acyl chain lengthon rates of spontaneous phospholipid transfer. Increasing the acyl chainlength to 18 carbons (DSPE-PEG₂₀₀₀, FIG. 12A) extended the lag in fusioneven further and, after 24 hours, the level was only 20% of maximum.

[0260] A second factor that affects the rate of spontaneous transfer ofphospholipids between bilayers is the degree of saturation orunsaturation of the acyl chains. The rate of fusion of LUVs containing 2mol % DOPE-PEG₂₀₀₀ is shown in FIG. 12B. The presence of a double bondincreased the rate of recovery of fusogenic activity in the presence ofa sink for the DOPE-PEG₂₀₀₀ over that of the corresponding saturatedspecies (DSPE-PEG₂₀₀₀, FIG. 12A). The rate of fusion was similar to thatseen with DPPE-PEG₂₀₀₀. FIG. 12B also shows the rate of fusion obtainedwhen the neutral PEG-lipid species, egg ceramide-PEG₂₀₀₀ was used. Therate was somewhat faster than observed with DPPE-PEG₂₀₀₀. Althoughdifferences in the interaction of the two lipid anchors with neighboringphospholipids in the bilayer make direct comparison of interbilayertransfer rates and, hence, fusion difficult, it appears that thepresence of a negative charge on the conjugate (PE-PEG) is not requiredfor desorption of the conjugate from negatively charged bilayers.

[0261] j. Effect of PEG Molecular Weight on Fusogenic Activity

[0262] The presence of PEG conjugated to the liposome surface results ina steric barrier that inhibits close bilayer apposition and subsequentfusion. The magnitude of the barrier should increase with increasing PEGmolecular weight. When DMPE-PEG₅₀₀₀ was incorporated into PE:PS (1:1)LUVS, a concentration dependent inhibition of fusion was observed (FIG.13A). The results are similar to those obtained with DMPE-PEG₂₀₀₀ (FIG.9), except that only 1 mol % DMPE-PEG₅₀₀₀ was required to completelyinhibit fusion compared to 2 mol % DMPE-PEG₂₀₀₀.

[0263]FIG. 13B shows the effect of varying acyl chain composition of thelarger PEG-lipid conjugate on fusion. Interestingly, the rates of fusionobserved with 1 mol % PE-PEG₅₀₀₀ were similar to those with 2 mol %PE-PEG₂₀₀₀. The concentrations used were those shown to be sufficient tocompletely inhibit fusion (cf., FIG. 9 and FIG. 13A). It was thoughtthat the larger PEG group would increase the rate of interbilayertransfer of the conjugate and, hence, the rate of fusion. However, thiswas not the case. To examine this aspect further, the rates of fusionunder conditions where the initial surface density of ethylene glycolgroups was similar were compared. FIG. 14 shows the fusion of PE:PS(1:1) LUVs containing 5 mol % DMPE-PEG₂₀₀₀ or 2 mol % DMPE-PEG₅₀₀₀ afteraddition of a sink for the PEG-lipid. The rates observed were verysimilar suggesting that factors other than loss of the steric barrier asa direct result of interbilayer transfer of the conjugate were involved.

[0264] k. Prgrammable Fusogenic Liposomes ComprisingDOPE:Cholesterol:DODAC:Ceramides

[0265] Fluorescently labelled liposomes were prepared in distilled waterfrom a mixture of DOPE and N,N-dioleoyl-N,N-dimethylammonium chloride(DODAC) at a molar ratio of 85:15. A three-fold excess of acceptorliposomes of the same composition, but containing no fluorescent probes,was added to labelled liposomes and fusion was initiated after 60 sec.by the addition of NaCl (FIG. 15). Fusion was highly dependent on ionicstrength. Little fusion was observed at 50 mM NaCl, but with increasingsalt concentration, the rate and extent of fusion increaseddramatically. At 300 mM NaCl fusion was so extensive that visibleaggregates occurred and these aggregates could not be maintained insuspension resulting in the apparent decrease in fluorescence seen inFIG. 15 for the 300 mM NaCl curve. Importantly, substantial fusion wasobserved at physiological salt concentration (150 mM).

[0266] As described above, the inclusion of 2 mol % PEG-lipid in PE:PSliposomes is sufficient to inhibit Ca²⁺-induced fusion. When 2 mol %DMPE-PEG₂₀₀₀ was included in DOPE:DODAC liposomes(DOPE:DODAC:DMPE-PEG₂₀₀₀, 83:15:2), the same inhibitory effect wasobserved (FIG. 16). However, unlike the PE:PS system, when theseliposomes were incubated for 1 hr. in the presence of a large excess ofPOPC liposomes, which acted as a sink for the PEG-PE, little, if any,fusion was observed. Since PEG-PEs are negatively charged thecomplementary charge, interaction with DODAC likely results in adramatic decrease in the rate of transfer out of the bilayer.

[0267] As an alternative bilayer stabilizing component, therefore, theability of a neutral PEG-lipid species, i.e., PEG-ceramide, to inhibitfusion in this system was examined. PEG-ceramides have similar bilayerstabilizing properties to PEG-PEs. For these studies, PEG₂₀₀₀ wasconjugated to ceramides of various fatty amide chain lengths through asuccinate linker. Liposomes prepared from DOPE:DODAC:(C8:0)ceramide-PEG₂₀₀₀ (83:15:2) did not fuse in the presence of 300 mM NaCl.However, when an excess of POPC liposomes was added, fusion occurredfairly rapidly (FIG. 17). Similar results were observed when cholesterolwas incorporated into the liposomes (DOPE:cholesterol:DODAC:(C8:0)ceramide-PEG₂₀₀₀, 38:45:15:2), although the rate of fusion was slowerthan with cholesterol-free liposomes (FIG. 17).

[0268] To determine if the rate of fusion in this system can becontrolled, the chain lengths of the fatty amide groups of thePEG-ceramides were varied. Using a (C14:0) ceramide-PEG₂₀₀₀, 50% maximalfusion was observed after approximately 6 hr (FIG. 18). This was adramatic increase over the rate with (C8:0) ceramide-PEG₂₀₀₀ shown inFIG. 18, where maximal fusion was achieved in about 40 minutes. The timefor 50% maximal fusion was increased to over 20 hr when eggceramide-PEG₂₀₀₀ was used. Ceramides derived from egg have a fatty amidechain length of predominantly 16:0 (approximately 78%), with smallamounts of longer saturated chains. FIG. 18 also shows an extended timecourse with DMPE-PEG₂₀₀₀. The limited extent of fusion (<20% of maximumat 21 hr) shows the dramatic effect that charge interaction can have onPEG-lipid transfer rates.

[0269] The rationale for using cationic liposomes is that complementarycharge interaction with anionic plasma membranes will promoteassociation and fusion of liposomes with cells in vivo. It is important,therefore, to confirm that not only will DOPE:DODAC liposomes fuse withmembranes carrying a negative charge, but that incorporation ofPEG-lipid conjugates prevents fusion in a programmable manner. Thisability is demonstrated in FIG. 19 which shows that liposomes composedof DOPE:cholesterol:DODAC, 40:45:15, fuse with negatively chargedliposomes and inclusion of a PEG-lipid conjugate in the cationicliposomes inhibits fusion. Fusion between DOPE:DODAC liposomes could beprevented when 2 mol % PEG-lipid was present in both fluorescentlylabelled and acceptor liposomes. When PEG-lipid was omitted from theacceptor liposomes, however, its concentration in the labelled vesicleshad to be increased to 4-5 mol % to block fusion between cationic andanionic liposomes.

[0270] Again, while PEG-lipids can inhibit fusion in this system, underconditions where the PEG-lipid can transfer out of the liposomes,fusogenic activity can be restored. FIG. 20 shows that this is, indeed,the case. Incubation of DOPE:cholesterol: DODAC:(C14:0) ceramide-PEG₂₀₀₀(36:45:15:4) liposomes with PE:PS liposomes, in the presence of excessPOPC:cholesterol (55:45) vesicles which act as a sink, results inrecovery of fusogenic activity. In the absence of a sink, a slow rate offusion is observed, indicating that a higher concentration of PEG-lipidis required to completely prevent fusion over longer periods.

[0271] While fusion between cationic and anionic liposomes provides agood model system, fusion in vivo is somewhat different. The acceptormembrane is not composed solely of lipid, but contains a highconcentration of proteins, many of which extend outward from the lipidbilayer and may interfere with fusion. Using erythrocyte ghosts as arepresentative membrane system, it was found that liposomes composed ofDOPE:cholesterol:DODAC (40:45:15) fuse with cellular membranes (see,FIG. 21). In addition, it was found that fusion in this system, likethose presented above, can also be inhibited using PEG-lipid conjugates.This results clearly establish the usefulness of these systems asprogrammable fusogenic carriers for intracellular drug delivery.

[0272] l. Programmed Fusion with Erythrocytes (RBCs)

[0273] LUVs composed of DOPE:cholesterol:DODAC (40:45:15) fused rapidlyand extensively with RBCs (FIG. 22, panels a and b). Prolongedincubation caused extensive lysis of the RBCs and numerous fluorescentlylabeled “ghosts” were formed. Incorporation of PEG-ceramide (C8:0) at 5mol % blocked fusion (FIG. 22, panels c and d) and this effect wasmaintained for up to 24 hr. This effect was somewhat surprising sincethe (C8:0) ceramide can exchange rapidly (i.e., within minutes) betweenliposomal membranes. It appears that either the RBCs cannot act as asink for the PEG-ceramide, or there were insufficient RBCs to removeenough PEG-ceramide to allow fusion. However, when an exogenous sink forthe PEG-ceramide was included, fusogenic activity was recovered withinminutes (FIG. 22, panels e and f).

[0274] When PEG-ceramides with longer fatty amide chains (i.e., C14:0 orC20:0) were used, there was little fusion over 24 hr, even in thepresence of an exogenous sink. This again was surprising as substantialfusion is observed over this time frame in liposomal systems when a sinkis present. It was thought that some non-specific interaction betweenthe sink (POPC/cholesterol) and the RBCs was occurring which hinderedthe ability of the POPC:cholesterol liposomes to absorb thePEG-ceramide. To overcome this, the fusogenic liposomes werepre-incubated with the sink before adding RBCS. FIG. 23 shows theresults obtained under these conditions using PEG-ceramide (C14:0). Nofusion was observed after pre-incubation of the fusogenic LUVs with thesink for 5 minutes prior to addition of RBCs (FIG. 23, panels a and b).However, after a 1 hr pre-incubation, some fusion with RBCs was observed(FIG. 23, panels c and d), suggesting that under these conditions thePEG-ceramide could transfer out of the liposomes and became fusogenic.With longer incubations (2 hrs.), the pattern of fluorescent labelingchanged. Rather than diffuse labeling of the RBC plasma membranes,extensive punctate fluorescence was observed (FIG. 23, panels e and f)and this pattern was maintained for up to 24 hr. The punctatefluorescence did not appear to be associated with cells and it mayrepresent fusion of fluorescent liposomes with the sink, althoughprevious fluorescent measurements of liposome-liposome fusion indicatedthat this did not occur to any appreciable extent. A second possibilityis that exchange of the fluorescent probe over the longer time coursesleads to labeling of the sink, although it seems unlikely that thiswould prevent fusion and labeling of the RBCS. When PEG-ceramide (C20:0)was used, there was no evidence for fusion after preincubation of LUVswith the sink for 5 min (FIG. 24, panels a and b), 1 hr (FIG. 24, panelsc and d), 2 hr (FIG. 24, panels e and f), or for up to 24 hr (resultsnot shown).

[0275] FIGS. 22-24 unequivocally establish that the liposomes of thepresent invention exhibit programmable fusion with intact cells.Firstly, liposomes composed of DOPE:cholesterol:DODAC (40:45:15) thatcontain no PEG-lipid fuse rapidly and extensively with RBCs. Secondly,when the liposomes contain 5 mol % PEG-lipid fusion is blockedregardless of the composition of the lipid anchor. Thirdly, in thepresence of a sink to which the PEG-lipid can transfer, fusogenicactivity can be restored at a rate that is dependent on the nature ofthe lipid anchor. Although exchange leading to fusion could not bedemonstrated when the PEG-ceramide (C20:0) was used, it is believed thisis a problem with the assay rather than a lack of fusogenic potential.In vivo there would be an almost infinite sink for PEG-lipid exchange.

[0276] m. Inhibition of Transmembrane Carrier System (TCS) Fusion byPEG₂₀₀₀-Ceramide (C14:0) and PEG₂₀₀₀-DMPE

[0277] TCS composed of 1,2-dioleoyl-3-phosphatidylethanolamine (DOPE),N,N-dioleoyl-N,N-dimethylammoniumchloride (DODAC), the fluorophoresN-(7-nitro-2-1,3-benzoxadiazol-4-yl)-1,2-dioleoyl-sn-phosphatidylethanolamine(NBD-PE) and N-(lissamine rhodamine Bsulfonyl)-1,2-dioleoyl-sn-phosphatidylethanolamine (Rh-PE), and eitherPEG₂₀₀₀-Ceramide (C14:0) or PEG₂₀₀₀-DMPE were prepared by extrusionthrough 100 nm diameter polycarbonate filters (Hope, M. J., et al., P.R. Biochim. Biophys. Acta, 812:55-65 (1985)). TCS contained 0.5 mol %NBD-PE and 0.5 mol % Rh-PE and either DOPE:DODAC:PEG₂₀₀₀-DMPE (80:15:5mol %) or DOPE:DODAC:PEG₂₀₀₀-Ceramide (C14:0) (80:15:5 mol %).Fluorescently labelled liposomes were incubated at 37° C. in 20 mMHEPES, 150 mM NaCl, pH 7.4 (HBS) with a three-fold excess of liposomescomposed of DOPE:POPS (85:15 mol %). POPC liposomes were added at10-fold the concentration of the fluorescently labelled liposomes andlipid mixing was assayed by the method of Struck, D. K., et al.(Biochemistry, 20:4093-4099 (1981)). The excitation wavelength used was465 nm and an emission filter placed at 530 nm minimized intensity dueto scattered light. Rates and extents of fusion were followed bymonitoring the increase in NBD fluorescence intensity at a wavelength of535 nm over time. Percent maximum fusion was determined from therelationship Fusion (% max)(t)=(F(t)-F_(o))/(F₂₈-F_(o)), where F_(o) isthe initial NBD fluorescence intensity at time zero, F(t) is theintensity at time t and F₂₈ is the maximum achievable fluorescenceintensity under conditions of complete lipid mixing of fluorescentlylabelled and DOPC:POPS liposomes (Bailey, A. L., et al., P. R.Biochemistry, 33:12573-12580 (1994)). FIG. 25 illustrates considerablemixing of DOPE/DODAC/PEG₂₀₀₀-Ceramide (C14:0) with DOPC:POPS compared tothat of DOPE/DODAC/PEG₂₀₀₀-DMPE with DOPC:POPS, suggesting that thePEG₂₀₀₀-DMPE is only minimally removed from the TCS. This result isattributed to the electrostatic interaction between the anionicPEG₂₀₀₀-DMPE and cationic DODAC which effectively decreases the monomerconcentration of the PEG₂₀₀₀-DMPE in aqueous solution.

[0278] n. In vivo Stabilization of Liposomes Containing Cationic Lipidsusing Amphiphatic Bilayer Stabilizing Components

[0279] The ability of a series of bilayer stabilizing components (e.g.,PEG-modified lipids) to stabilize fusogenic liposomes containing acationic lipid in vivo were examined in this study. A freeze-fractureelectron microscope analysis of liposomes composed ofdioleoylphosphatidylethanolamine (DOPE) andN,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) showed that inclusionof a bilayer stabilizing component, e.g., PEG-DSPE and PEG-Ceramide,effectively prevented liposome aggregation in the presence of mouseserum. Biodistribution of fusogenic liposomes composed of DOPE andDODAC, additionally containing a bilayer stabilizing component (i.e., anamphiphatic polyethyleneglycol (PEG) derivative), were then examined inmice using ³H-labelled cholesterylhexadecylether as a lipid marker.Bilayer stabilizing components included PEG-DSPE and variousPEG-Ceramide (PEG-Cer) with different acyl chain length ranging from C8to C24. DOPE/DODAC liposomes (85:15, mol/mol) were shown to be clearedrapidly from the blood and accumulate exclusively in the liver.Inclusion of a bilayer stabilizing component at 5.0 mol % of the lipidmixture resulted in increased liposome levels remaining in the blood andconcomitantly decreased accumulation in the liver. Among the variousbilayer stabilizing components, PEG-DSPE shows the highest activity inprolonging the circulation time of DOPE/DODAC liposomes. The activity ofPEG-Ceramide is directly proportional to the acyl chain length: thelonger the acyl chain, the higher the activity. The activity ofPEG-Ceramide (C20) exhibiting the optimal acyl chain length depends onits concentration of the lipid mixture, with the maximal circulationtime obtained-at 30 mol % of the lipid mixture. While inclusion ofbilayer stabilizing components in the lipid composition generallyresults in increased circulation time of DOPE/DODAC liposomes, thepresence of a cationic lipid, DODAC, appeared to promote their rapidclearance from the blood.

[0280] The preparations and uses of DODAC liposomes are disclosed inU.S. patent application Ser. No. 08/316,399, filed Sep. 30, 1994, theteachings of which are incorporated herein by reference.

[0281] i. Materials and Methods

[0282] aa. Liposome Preparation

[0283] Small unilamellar liposomes composed of DOPE and DODAC andbilayer stabilizing components at various ratios were prepared by theextrusion method. Briefly, the solvent-free lipid mixture containing³H-labelled CHE, as a nonexchangeable and nonmetabolizable lipid marker,was hydrated with distilled water overnight. Normally, the liposomesuspension (5 mg lipid per ml) was extruded, at room temperature, 10times through stacked Nuclepore membranes (0.1 μm pore size) using anextrusion device obtained from Lipex Biomembranes, Inc. to generateliposomes with homogeneous size distributions. Liposome size wasdetermined by quasi-elastic light scattering using a particle sizer andexpressed as average diameter with standard deviation (SD).

[0284] bb. Liposome Biodistribution Study

[0285]³H-labelled liposomes with various lipid compositions wereinjected i.v. into female CD-1 mice (8-10 weeks old) at a dose of 1.0 mglipid per mouse in 0.2 ml of distilled water. At specified timeintervals, mice were killed by overexposure to carbon dioxide, and bloodwas collected via cardiac puncture in 1.5-ml microcentrifuge tubes andcentrifuged (12000 rpm, 2 min, 4° C.) to pellet blood cells. Majororgans, including the spleen, liver, lung, heart, and kidney, werecollected, weighed, and homogenized in distilled water. Fractions of theplasma and tissue homogenates were transferred to glass scintillationvials, solubilized with Solvable (NEN) at 50° C. according to themanufacturer's instructions, decolored with hydrogen peroxide, andanalyzed for ³H radioactivity in scintillation fluid in a Beckmancounter. Data were expressed as percentages of the total injected doseof ³H-labelled liposomes in each organ. Levels of liposomes in theplasma were determined by assuming that the plasma volume of a mouse is5.0% of the total body weight.

[0286] ii. Results and Discussion

[0287] aa. Freeze-Fracture Electron Microscopic Studies

[0288] Liposomes composed of DOPE/DODAC (85:15, mol/mol),DOPE/DODAC/PEG-Ceramide (C20) (80:15:5, mol/mol), andDOPE/DODAC/PEG-DSPE (80:15:5, mol/mol) were prepared by the extrusionmethod and had similar average diameters (100 nm). Freeze-fractureelectron micrographs of the three liposomal formulations showedunilamellar liposomes with relatively narrow size ranges. However,preincubation of DOPE/DODAC liposomes in 50% mouse serum at 37° C. for30 minutes resulted in their massive aggregations. On the other hand,both DOPE/DODAC/PEG-Ceramide (C20) and DOPE/DODAC/PEG-DSPE liposomes didnot show any aggregation when these liposomes were pretreated with mouseserum. Thus, these results show the effectiveness of the bilayerstabilizing components in preventing serum-induced rapid aggregations ofDOPE/DODAC liposomes.

[0289] bb. Biodistribution of DOPE/DODAC Liposomes Containing Bilayer

[0290] Stabilizing Components, i.e., Amphiphatic PEG Derivatives

[0291] DOPE/DODAC liposomes with or without bilayer stabilizingcomponents were prepared to include ³H-labelledcholesterolhexadecylether as a lipid marker, and their biodistributionwas examined in mice at 1 hour after injection. Liposomes tested in thisstudy were composed of DOPE/DODAC (85:15, mol/mol),DOPE/DODAC/PEG-Ceramide (80:15:5, mol/mol), and DOPE/DODAC/PEG-DSPE(80:15:5, mol/mol). To also examine the effect of the hydrophobic anchoron biodistribution of liposomes, various PEG-Ceramide derivatives withdifferent acyl chain lengths were used. These liposomal formulations hadsimilar average diameters, ranging from 89 to 103 nm. Table II belowshows levels of liposomes in the blood, spleen, liver, lung, heart, andkidney, together with respective blood/liver ratios. DOPE/DODACliposomes were shown to be cleared rapidly from the blood and accumulatepredominantly in the liver with the blood/liver ratio of approximately0.01. Inclusion of bilayer stabilizing components at 5.0 mol % in thelipid composition resulted in their increased blood levels andaccordingly decreased liver accumulation to different degrees.DOPE/DODAC/PEG-DSPE liposomes showed the highest blood level (about 59%)and the lowest liver accumulation (about 35%) with the blood/liver ratioof approximately 1.7 at 1 hour after injection. Among variousPEG-Ceramide derivatives with different acyl chain lengths, PEG-Ceramide(C20)-containing liposomes showed the highest blood level (about 30%)with the blood/liver ratio of approximately 0.58, while PEG-Ceramide(C8)-containing liposomes showed a lower blood level (about 6%) with theblood/liver ratio of approximately 0.1. It appeared that, amongdifferent PEG-Ceramide derivatives, the activity in increasing the bloodlevel of liposomes is directly proportional to the acyl chain length ofceramide; the longer the acyl chain length, the greater the activity. Italso appeared that the optimal derivative for increasing the blood levelof liposomes is PEG-Ceramide (C20).

[0292] cc. Optimization of DOPE/DODAC Liposomes for ProlongedCirculation Times

[0293] The effect of increasing concentrations of PEG-Ceramide (C20) inthe lipid composition on biodistribution of DOPE/DODAC liposomes wasexamined. PEG-Ceramide (C20) was included in DOPE/DODAC liposomes atincreasing concentrations (0-30 mol %) in the lipid composition, whilethe concentration of DODAC was kept at 15 mol % of the lipid mixture.Liposomes were prepared by the extrusion method and had similar averagediameters ranging from 102 nm to 114 nm. Liposomes were injected i.v.into mice, and biodistribution was examined at 1 hour after injections.FIG. 26 shows the liposome level in the blood and liver at 1 hour afterinjections as a function of the PEG-Ceramide (C20) concentration.Clearly, increasing the concentration of PEG-Ceramide in the lipidcomposition resulted in progressive increase in liposome levels in theblood, accompanied by decreased accumulation in the liver. The highestblood level (about 84% at 1 hour after injection) was obtained forDOPE/DODAC/PEG-Ceramide (C20) (55:15:30, mol/mol) showing theblood/liver ratio of about 6.5.

[0294] The effect of increasing concentrations of DODAC on thebiodistribution of DOPE/DODAC liposomes also was examined. DOPE/DODACliposomes containing either 10 mol % or 30 mol % PEG-Ceramide (C20) andvarious concentrations (15, 30, 50 mol %) were prepared by the extrusionmethod and had similar average diameters ranging from 103 to 114 nm.Biodistribution was examined at 1 hour after injections, and expressedas percentages-of liposomes in the blood as a function of the DODACconcentration (FIG. 27). As shown in FIG. 27, increasing DODACconcentrations in the lipid composition resulted in decreased levels inthe blood for both liposomal formulations. Thus, the presence of acationic lipid, DODAC, in the lipid composition results in rapidclearance from the blood. Also, shown in FIG. 27 is that such a DODACeffect can be counteracted by increasing the concentration ofPEG-Ceramide (C20) in the lipid composition.

[0295]FIG. 28 shows time-dependent clearances of DOPE/DODAC liposomeswith or without PEG-Ceramide from the blood. Only a small fraction ofinjected DOPE/DODAC liposomes remained in the blood, while increasingthe concentration of PEG-Ceramide (C20) in the lipid compositionresulted in prolonged circulation times in the blood. Estimatedhalf-lives in the α-phase for DOPE/DODAC/PEG-Ceramide (C20) (75:15:10,mol/mol) and DOPE/DODAC/PEG-Ceramide (C20) (55:15:30, mol/mol) were <1hour and 5 hours, respectively.

[0296] iii. Conclusions

[0297] The above studies indicate that there are several levels at whichbiodistribution of fusogenic liposomes containing a cationic lipid canbe controlled by inclusion of bilayer stabilizing components. Data inTable II shows that the hydrophobic anchor of the bilayer stabilizingcomponents has an important role in determining biodistribution ofDOPE/DODAC liposomes. Studies using various PEG-Ceramide derivativeswith different acyl chain lengths showed that the longer the acyl chainlength of PEG-Ceramide, the greater the activity in prolonging thecirculation time of DOPE/DODAC liposomes. These results are consistentwith the rate at which the bilayer stabilizing components dissociatefrom the liposome membrane being directly proportional to the size ofthe hydrophobic anchor. Accordingly, PEG-Ceramide derivatives with alonger acyl chain can have stronger interactions with other acyl chainsin the liposome membrane and exhibit a reduced rate of dissociation fromthe liposome membrane, resulting in stabizzation of DOPE/DODAC liposomesfor a prolonged period of time and thus their prolonged circulation timein the blood.

[0298] In addition to the hydrophobic anchor of the bilayer stabilizingcomponents, the concentration of the bilayer stabilizing components inthe lipid membrane can also be used to control in vivo behavior ofDOPE/DODAC liposomes. Data in FIG. 26 show that increasing theconcentration of PEG-Ceramide (C20) in the lipid composition resulted inincreased liposome levels in the blood. The optimal concentration ofPEG-Ceramide (C20) in the lipid composition was found to be 30 mol % ofthe lipid mixture. It appeared that the circulation time ofDOPE/DODAC/PEG-Ceramide (C20) liposomes is determined by the relativeconcentrations of two lipid compositions, DODAC and PEG-Ceramide,exhibiting opposite effects on liposome biodistribution. While bilayerstabilizing components exhibit the ability to prolong the circulationtime of liposomes in the blood, a cationic lipid, DODAC, exhibits theability to facilitate liposome clearance from the blood. Thus, for themaximal circulation time in the blood, an appropriate concentration of abilayer stabilizing component and a minimal concentration of DODACshould be used. It should be noted, however, that an optimal liposomeformulation for the prolonged circulation time in the blood is notnecessarily the one suitable for an intended application in delivery ofcertain therapeutic agents. Both pharmacokinetic and pharmacodynamicaspects of fusogenic liposomes should be examined for differentapplications using different therapeutic agents. TABLE II Effect ofAmphipathic PEG Derivatives on Biodistribution of DOPE/DODAC Liposomes %injected dose PEG- Average Derivative Diameter (nm) Blood Liver SpleenLung Heart Kidney Total Blood/Liver None 103 (29)  0.8 (0.4) 64.4 (2.0)3.1 (1.8) 1.2 (0.2) 0.2 (0.0) 0.3 (0.0)  70.0 (1.4) 0.012 PEG-DSPE  95(26) 59.1 (8.2) 34.7 (2.1) 2.9 (0.1) 1.9 (0.8) 1.7 (0.4) 1.2 (0.5) 101.4(6.1) 1.703 PEG-Cer (C8)  89 (24)  6.5 (1.9) 62.8 (3.4) 4.2 (1.0) 0.5(0.3) 0.3 (0.1) 0.3 (0.1)  74.6 (5.1) 0.104 PEG-Cer (C14)  93 (25)  5.9(0.5) 55.9 (1.0) 3.3 (0.2) 0.1 (0.0) 0.1 (0.0) 0.1 (0.0)  65.4 (1.6)0.106 PEG-Cer (C16)  93 (24) 13.9 (2.1) 57.5 (2.0) 2.6 (0.1) 0.0 (0.0)0.2 (0.1) 0.0 (0.0)  74.3 (4.0) 0.242 PEG-Cer (C20) 101 (24) 29.8 (4.8)51.0 (2.2) 1.9 (0.2) 0.0 (0.0) 0.3 (0.1) 0.0 (0.0)  82.8 (2.8) 0.584PEG-Cer (C24)  92 (28) 26.7 (0.8) 46.7 (7.6) 5.7 (1.2) 1.0 (0.2) 0.9(0.2) 0.4 (0.1)  81.5 (4.1) 0.572

[0299] B. Examples Relating to the Fusogenic Liposomes ContainingLipopeptides

[0300] 1. MATERIALS AND GENERAL METHODS

[0301] a. Lipids and Chemicals

[0302] Crude peptide was obtained from the laboratory of Dr. IanClark-Lewis, Biomedical Research Laboratory, University of BritishColumbia. Subsequently, purified peptide was purchased from MultipleSynthesis (CA). 1,2-Distearoyl-sn-glycerol (DSG),1-Palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC, eggphosphatidylcholine (EPC),N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-4-yl)-1,2-dioleoyl-sn-phosphatidylethanolamine(NBD-PE), and N-(lissamine rhodamine Bsulphonyl)-1,2-dioleoyl-sn-phosphatidylethanolamine (Rh-PE) weresupplied by Avanti Polar Lipids (Alabaster, Ala.).Aminonaphthalenetrisulphonic acid (ANTS) and p-xylylenebis-(pyridinium)bromide (DPX) were purchased from Molecular Probes (Eugene, Oreg.).Succinic anhydride, 4-dimethylaminopyridine, N-hydroxysuccinimide,dicyclohexylcarbodiimide, cholesterol and all buffers were purchasedfrom Sigma Chemical Co. (St. Louis, Mo.). HPLC-grade organic solventsand miscellaneous chemicals were supplied by Fisher Scientific.

[0303] b. Preparation of AcE4K and Lipo-AcE4K

[0304] After lyophilization, the peptide was purified by reverse-phaseHPLC on a Synchropak (Synchrom, Inc.) C8 semi-preparative HPLC columnusing a 40%-70% linear gradient of acetonitrile in water (0.1% TFA) witha flow rate of 6 ml/minute over 20 minutes. The peptide elutes atapproximately 55% acetonitrile. Composition and purity of the peptidewere verified by amino acid analysis, mass spectrometry and HPLC. Puritywas estimated to be greater than 95%.

[0305] The synthesis of the lipopeptide is illustrated in FIG. 40. Onegram of 1,2-distearoyl-sn-glycerol (1.6 mmol) (I), 0.2 g succinicanhydride (2 mmol), and 0.24 g 4-dimethylaminopyridine (2 mmol) weredissolved in 10 ml of CH₂Cl₂ and stirred at room temperature for onehour. The resulting acid (2) was isolated by removing solvent by rotaryevaporation followed by purification by silica gel chromatography using10% ethyl acetate in hexane as eluant. Two hundred milligrams of thismaterial (0.28 mmol) and 32 mg of N-hydroxysuccinimide (0.29 mmol) weredissolved in 5 ml of CH₂Cl₂ and 57 mg of 1,3-dicyclohexylcarbodimide(0.28 mmol) was added with stirring. The reaction was allowed to proceedfor one hour at room temperature after which the mixture was filtered toremove precipitate, and the solvent was removed by rotary evaporationyielding the activated lipid (3). A mixture of 5.6 mg of the peptideAcE4K (2.5 μmol), 4.1 mg of 3 (5.0 μmol) and 15 mg of triethylamine in 1ml of dimethylsulfoxide (DMSO) was heated to 65° C. to achieveco-dissolution of the lipid and peptide and incubated for one hour.After cooling, the lipopeptide (4) was precipitated by the addition of 5ml of diethyl ether and centrifuged at 2000 rpm for 5 minutes. Thepellet was washed three times with 2 ml of diethyl ether repeating thecentrifugation with each wash. The lipopeptide was dried under vacuumand its identity was confirmed by mass spectrometry. Purity asdetermined by peptide-to-lipid ratio using ¹H-NMR was found to begreater than 95%.

[0306] c. Preparation of Liposomes

[0307] Chloroform solutions of lipids were dried by vortex mixing undernitrogen followed by the removal of residual solvent under high vacuumfor 1 hour. When lipopeptide was incorporated into the liposomepreparations, it was added to the dried lipids as a 1 mM solution inDMSO along with an equal volume of benzene-methanol (95:5) prior tofreeze-drying for 5 hours. Lipids were hydrated with appropriate buffersto concentrations ranging from 5 to 20 mM lipid. Five freeze-thaw cycleswere used to ensure homogeneous mixture of the multilamellar vesicle(MLV) suspensions. The MLVs were extruded 10 times through two 100 nmpore-size polycarbonate filters (Costar, Cambridge, Mass.) to producelarge unilamellar vesicles (LUVs). Lipid concentrations were determinedby phosphate assay as described previously (see, e.g., Bartlett, G. R.,J. Biol. Chem., 234:466-68 (1959)). Depending on the lipid formulation,the mean diameter of the LUVs ranged from 100 to 135 nm as measured byquasi-elastic light scattering.

[0308] d. Circular Dichroism

[0309] Differences in the secondary structure of AcE4K and Lipo-AcE4K asa function of pH were investigated by CD spectropolarimetry. A solutionof AcE4K was initially dissolved in 10 mM phosphate buffer, pH 7.5, at aconcentration of 0.5 mM. Subsequently, 200 μl samples were prepared bydiluting this stock to a peptide concentration of 25 μM in 10 mMphosphate buffer at either pH 5.0 or pH 7.5. CD spectra over wavelengthsof 200 to 250 nm were recorded on a Jasco J720 Spectropolarimeter usinga 1 mm quartz cuvette and accumulations of five scans. To obtain spectrain the presence of lipid bilayers, POPC LUVs were used at aconcentration of 2.5 mM lipid (lipid/peptide ratio=100) prepared in 10mM phosphate buffer adjusted to either pH 7.5 or pH 5.0. Under theseconditions, the CD spectra of the peptide could be measured in thepresence of lipid bilayers with minimal difficulties arising fromabsorbance and scattering due to the lipid. The spectra obtained werecorrected by subtracting lipid or buffer signal, as appropriate.

[0310] For the lipopeptide, LUVs were prepared with 1% Lipo-AcE4K inPOPC at a phospholipid concentration of 2.5 mM in 10 mM phosphate bufferadjusted to either pH 5.0 or pH 7.5. CD spectra were obtained as above.The very low solubility of the lipopeptide prevents measurements ia theabsence of lipid.

[0311] e. Tryptophan Fluorescence

[0312] Tryptophan fluorescence spectra were recorded with an excitationwavelength of 280 nm over an emission range of 300 to 400 nm on a PerkinElmer LS50 fluorometer using a 1 cm quartz cuvette thermostatted at 25°C. For the free peptide, the aqueous stock solution was diluted to 100μM and 30 μl of this was added to 10 mM phosphate buffer at pH 7.5 or pH5.0, either with or without POPC LUVs (0.1 mM phospholipid,lipid/peptide ratio=100), for a total sample volume of 3 ml. Spectra ofthe lipopeptide incorporated into liposomes were obtained using theLipo-AcE4K/POPC LUVs described above diluted to 0.1 mM POPC, 1.0 μMLipo-AcE4K. The spectra were corrected by subtracting scans of phosphatebuffer or LUVs, as appropriate.

[0313] f. Preparation of Erythrocyte Membranes

[0314] Sealed erythrocyte ghosts were prepared by the method of Steckand Kane, supra (1974). Briefly, 4 ml of packed cells was washed 3 timeswith HEPES buffered saline (HBS: 5 mM HEPES, 150 mM NaCl, pH 7.5),centrifuging each for 5 minutes at 2000 rpm in a swinging-bucket rotor.Washed cells were diluted 2-fold with HBS, lysed in 300 ml of 5 mMHEPES, 1 mM MgCl₂, pH 7.5, and pelleted at 20,000 g for 20 minutes.Ghosts were removed from above the hard, protease-rich pellet andresuspended in 200 ml of HBS containing 1 mM MgCl₂. The suspension wasrepelleted and washed twice more and finally resuspended in 10 ml ofHBS. Phospholipid concentration was determined by phosphate assay. Theabsence of glyceraldehyde-3-phosphate dehydrogenase activity (Steck &Kant, supra, 1974) was used to confirm the formation of sealedright-side-out ghosts.

[0315] g. Lipid-Mixing Fusion Assays

[0316] The extent of membrane fusion as measured by lipid mixing in thepresence of AcE4K and Lipo-AcE4K was monitored by the decrease inresonance energy transfer (RET) resulting from dual fluorescent probedilution (Struck, et al., Biochemistry, 20:4093-4099 (1981)). LUVs of adesired lipid composition containing of 0.5 mol % of both NBD-PE andNBD-PE were prepared in HMA buffer (10 mM HEPES, 10 mM MES, 10 mM sodiumacetate, 100 mM NaCl), pH 7.5. AcE4K was added to labeled vesicles froma 1 mM aqueous solution at pH 7.5. Lipo-AcE4K was either included in thelipid preparation as described above or added to labeled vesicles from a1 mM solution in DMSO. Labeled vesicles were mixed with eitherunlabelled LUVs or erythrocyte ghosts in a lipid ratio of 1:3 at a totallipid concentration of 0.2 mM. Typically, 15 μl 10 mM labeled LUVs and45 μl of 10 mM unlabelled LUVs or 300 μl of erythrocyte ghosts were madeup to 3 ml in a 1 cm quartz cuvette with a HMA buffer, pH 7.5.Fluorescence was monitored at 25° C. over 5 minutes with excitation at465 nm, emission at 535 nm, and an emission cut-off filter at 530 mn.During the assays, 1 M HCl was added to decrease the pH to a desiredvalue. (HMA buffer has a linear pH response to acid volume over the pHrange 4.0 to 7.5.)

[0317] Each point in the lipid-mixing timecourse was normalized bysubtracting the fluorescence of a comparable assay lacking unlabelledvesicles (F₀) and dividing by the fluorescence achieved by infiniteprobe dilution determined by the addition of 25 μl of 100 mM TritonX-100 (F_(max)). The percent change in fluorescence was calculated as${\% \quad \frac{\Delta \quad F}{\Delta \quad F_{\max}}} = {100 \times \left( \frac{F - F_{0}}{F_{\max} - F_{0}} \right)}$

[0318] for each point in the timecourse. Complete lipid mixing, asdetermined by a liposome preparation corresponding to a 1:3 ratio oflabeled to unlabelled vesicles, gives a value of ΔF/ΔF_(max) ofapproximately 80% under these conditions. Reported results were notcorrected by this factor.

[0319] h. Exchange of Lipo-AcE4K Between Membranes

[0320] POPC MLVs were prepared in HMA buffer at pH 7.5 as describedabove and pelleted at 12,000 rpm on a benchtop centrifuge at 5° C. Thepellet was resuspended in HMA buffer and repelleted, and this procedurewas repeated for three washings to ensure removal of any small lipidvesicles prior to determination of lipid concentration by phosphateassay. Thirty microlitres of POPC LUVs (10 mM lipid) was diluted in 1.09ml of HMA buffer, pH 7.5, and 7.5 μl of 2 mM Lipo-AcE4K was added fromDMSO stock. This preparation results in the incorporation of 10 mol % ofthe lipopeptide into the outer monolayer of the LUVs. After a 5 minutepre-incubation at 25° C., 375 μl of 12.5 mM POPC MLVs were added as asink for lipopeptide exchange. This 60-fold lipid excess represents a3-fold excess of available sink, assuming 5% of the MLV lipid is exposedon the outermost monolayer. Following a five minute incubation at 25°C., the MLVs were pelleted as above and the peptide and lipid content(phosphate assay) of the supernatant was determined. A micro-BCA assaykit as provided by Pierce Chemical Co. was used with the providedprocedure to analyze for peptide. The results were compared to controlswithout MLVs or without Lipo-ACE4K.

[0321] i. Contents Mixing and Leakage

[0322] Liposomes of a desired composition were prepared containingeither 25 mM ANTS in HMA buffer, 100 mM DPX in HMA buffer, or 6 mM ANTSplus 75 mM DPX (ANTS-DPX), at pH 7.5 as described above. External bufferwas exchanged with HMA buffer on Sephadex G-25 columns prior to dilutingto 10 mM lipid. To assay for contents mixing, 15 μl of the ANTSpreparation on 45 μl of DPX liposomes were combined in 3 ml of HMAbuffer. ANTS fluorescence was monitored over 5 minutes with the additionof 15 μl of 1 M HCl at 30 seconds to decrease the pH to 5.0. Excitationand emission wavelengths were 360 nm and 530 nm, respectively, and a 490nm cut-off filter was used. Maximum quenching and zero leakage wasdetermined by the fluorescence of the preparation containing both ANTSand DPX, and zero quenching was measured using only ANTS liposomes, bothprior to the addition of acid. Leakage was quantified by comparing themaximum quenching result (0% leakage) with a similar assay to which 25μl of 100 mM Triton X-100 was added (100% leakage).

[0323] j. Freeze-Fracture Electron Microscopy

[0324] LUVs consisting of 10 mol % Lipo-AcE4K in EPC/Chol (55:45) wereprepared in HMA buffer, pH 7.5, at a total lipid concentration of 5 mM.A sample at pH 5.0 was prepared by adding 1.5 μl of 1 M HCl to 100 μl ofliposomes. After 5 minute incubations at 25° C., samples at each pH weremixed 1:1 with glycerol and quickly frozen. Platinum/carbon replicaswere prepared as described previously (Fisher & Branton, 1974). EPC/Chol(55:45) liposomes at pH 7.5 and 5.0 were used as controls.

[0325] k. Fluorescence Microscopy

[0326] To confirm lipid mixing of Lipo-AcE4K LUVs with erythrocytemembranes, the appearance of Rh-PE fluorescence in the erythrocytemembranes upon acidification was demonstrated. The dual-labeled liposomepreparation as described above for the lipid mixing assay was used. Onehundred microlitres of 2.5 mM LUVs and 10 μl of 0.25 mM Lipo-AcE4K inDMSO were added to 615 μl of HMA buffer, pH 7.5. After a 5 minutepreincubation, a 3-fold lipid excess of erythrocyte ghosts (250 μl of 3mM lipid) was added. This mixture contained 1 mM total lipid with 10 mol% Lipo-AcE4K incorporated into the outer monolayers of the LUVs, or fivetimes the concentrations used in the lipid mixing assays. A 5 μl aliquotwas removed prior to acidification with 15 μl of 1 M HCl, reducing thepH to 5.0. Samples at each pH were inspected by confocal microscopyusing both phase-contrast and fluorescence techniques.

[0327] 2. EXPERIMENTAL RESULTS

[0328] a. Solubilities of AcE4K and Lipo-AcE4K

[0329] The highly hydrophobic nature and low solubility of natural viralfusion peptides is problematic in studying their interactions with lipidvesicles. The peptide AcE4K was soluble in aqueous solutions at pH 7.5at concentrations up to 10 mM and highly soluble in DMSO. The use of theC-terminal lysine residue to couple the peptide to the lipid anchor,rather than the more commonly used cyteine-thioether chemistry, overcameearlier difficulties which existed with purifying the correspondingC-terminal cysteine peptide. The lipopeptide Lipo-AcE4K was soluble onlyin DMSO and was added to assays from a 2 mM stock solution such that theamount of organic solvent was less than one percent by volume.

[0330] b. Circular Dichroism and Tryptophan Fluorescence

[0331] Differences in the secondary structure of AcE4K and Lipo-AcE4K asa function of pH were investigated by CD spectropolarimetry. CD spectraof AcE4K and Lipo-AcE4K are given in FIG. 41. The behavior of thelipopeptide as a function of pH is markedly different from the freepeptide. AcE4K has a random coil structure at neutral pH either in thepresence or absence of POPC LUVs (FIG. 41(A)). At pH 5.0, the randomcoil signal persists in the absence of lipid membranes. However, in thepresence of POPC LUVs at pH 5.0, AcE4K adopts a highly α-helicalstructure, characterized by the signal minima at 208 nm and 222 nm. Thisresult suggests that AcE4K can exist as a amphipathic helix, much likethe structure given in FIG. 39, and that it does so only uponneutralization of its acidic residues and in the presence of lipidbilayers. In contrast, the structure of the peptide in Lipo-AcE4K doesnot appear to be affected by pH (FIG. 41(B)). At pH 7.5, the lipopeptidealready exists in a partly α-helical conformation and the CD spectrum isnot changed at pH 5.0. This difference in behavior for the peptide inits free and lipid-coupled form are surprising, but these results do notprovide any information on the degree of interactions of the peptideswith the lipid bilayer or their effects on membrane stability.

[0332] The penetration of peptides bearing tryptophan or tyrosineresidues into lipid bilayers can be monitored by the fluorescenceemission spectra of these amino acid residues. Collisional quenching offluorescence caused by water when the residues are exposed to aqueousmedium is reduced upon membrane penetration. This is accompanied by ablue shift in the maximum of fluorescence emission resulting from thereduced polarity of the medium. AcE4K contains a single tryptophanresidue at position 14 and has no tyrosine residues. The fluorescencespectrum of tryptophan-14 can, therefore, be used as a measure ofmembrane penetration. The spectral of AcE4K at pH 7.5 and 5.0 in thepresence and absence of POPC LUVs are given in FIG. 42(A). Nodifferences are observed, except for the sample at pH 5.0 in thepresence of vesicles which exhibits a slight blue shift of λ_(max) and asignificant increase in intensity at shorter wavelengths. In contrast tothis, the spectra for Lipo-AcE4K in POPC vesicles shown in FIG. 42(B)indicate that, even at pH 7.5, the tryptophan residue is somewhatprotected from the aqueous medium having a λ_(max) of 340 nm compared to355 nm for the free peptide. At pH 5.0, the λ_(max) is further reducedto 332 nm. This result suggests that, while no structural changes in thepeptide were observed in the CD spectrum, Lipo-AcE4K penetrates furtherinto the lipid bilayer upon neutralization of the acidic residues.However, the observed changes could also arise from the protection ofthe tryptophan residue from the aqueous medium through the formation ofoligomeric complexes of the lipopeptide within the membrane.

[0333] c. Fusion of Liposomes Induced by AcE4K and Lipo-AcE4K

[0334] The destabilization of membranes accompanying the observedchanges in peptide structure and membrane penetration was studied bymonitoring the fusion of lipid vesicles as measured by lipid mixing,contents mixing and leakage. Membrane fusion was expected to depend onthe extent of neutralization of the acidic residues of Lipo-AcE4K and toincrease with decreasing pH.

[0335] The effects of peptide structure and membrane penetration as afunction of pH on the stability of liposomal membranes and fusion ofliposomes were monitored by the loss of RET between the fluorescentlylabeled lipids, NBD-PE and Rh-PE. Vesicles containing both probes aremixed with unlabeled vesicles, and membrane fusion results in probedilution and increased NBD-PE fluorescence. Exchange of the labeledlipids does not occur over the duration of these experiments, even inhighly aggregated systems (Hoekstra, et al., Biochemistry, 21:6097-6103(1982)), and fluorescence increases only upon mixing of membrane lipids.

[0336] Initially, we looked at fusion of POPC LUVs with 5 mol %Lipo-AcE4K added to the preformed vesicles from a DMSO stock solution.Lipid mixing fluorescence timecourses upon acidification to PH's between7.0 and 4.0 are shown in FIGS. 45A and 45B. No lipid mixing was observedabove pH 6.0. However, there was a substantial increse in mixing betweenpH 5.75 and 5.5. These changes may have physiological importance, inthat the pH of the endosomal interior falls in the range of 5 to 6. AtpH 5.0 an initial rapid increase in NBD-PE fluorescence levels off overone to two minutes, indicating a transient destabilization of themembrane. Further decreases in pH give even greater lipid mixing, and atpH 4.0 the initial rapid increase in fluorescence is followed by aslower rise over several minutes. Similar experiments in which the freepeptide, AcE4K, was added to POPC vesicles also gave lipid mixing, butat levels about half of those observed for the lipopeptide (data notshown).

[0337] The effect of Lipo-AcE4K concentration in the outer monolayer ofPOPC vesicles on the degree of lipid mixing at pH 5.0 is shown in FIG.44A. Small but significant increases in NBD-PE fluorescence is observedwith as little as 1 mol % Lipo-AcE4K, and the level of mixing achievedincreases up to 10 mol %, the maximum level assayed. In all cases, theobserved increase in fluorescence is complete within 1 or 2 minutes. Thetransient nature of the lipid mixing in all of these cases suggests theloss of destabilizing capability of the lipopeptide, perhaps throughconformational changes not detectable by CD experiments or through theformation of oligomeric complexes.

[0338] d. Contents Mixing and Leakage

[0339] To determine whether the pH-induced destabilization of membranescontaining Lipo-AcE4K corresponded to fusion events with contents mixingsubsequent to the observed lipid mixing, the ANTS-DPX contents mixingassay was used. In this assay, fusion between a liposome populationcontaining the fluorescent marker ANTS and a second populationcontaining the quencher DRX results in a loss of ANTS fluorescence. Theassay is not affected by moderately acidic conditions and candistinguish contents mixing from probe leakage, since the latter resultsin an insufficient concentration of DPX to provide quenching. Leakagewas separately determined by monitoring ANTS dequenching for liposomescontaining both ANTS and DPX.

[0340] For the range of Lipo-AcE4K concentrations used above (1 to 10mol %) in POPC vesicles, no contents mixing was observed. The ANTS-DPXassay revealed only the leakage of vesicle contents upon decreasing thepH to 5.0, and the extent of leakage observed corresponded to theconcentration of lipopeptide as shown in FIG. 44B. With 10 mol %Lipo-AcE4K in the outer monolayer, all of the probe leaked out withinone minute. At lower lipopeptide concentrations, most of the leakageoccurred within the first minute followed by much slower leakage. Thisbehavior corresponds to the transient rapid lipid mixing which waspreviously observed, and again suggests a rapid loss of destabilizingcapability for the lipopeptide.

[0341] The absence of contents mixing, the occurrence of rapid leakageand accompanying lipid mixing observed in these systems clearly do notdescribe the complete fusion process as achieved by the virus. Theresults are consistent with previous studies on the destabilization oflipid vesicles with viral fusion peptides. In no case has contentsmixing arising from a non-leaky fusion event been convincinglydemonstrated. However, the ability of the lipopeptide to transientlydestabilize lipid bilayers suggests that fusion peptides have afunctional role in destabilizing target membranes as well as ananchoring role in bringing fusing membranes into close apposition, andthat the entire fusion protein is required to give a complete fusionevent. This investigation was continued by looking at the ability ofLipo-AcE4K present in one membrane to destabilize synthetic andbiological target membranes which are otherwise pH stable.

[0342] Prior to investigating the fusion of Lipo-AcE4K-containingliposomes to stable vesicles lacking lipopeptide or to biologicalmembranes, it was necessary to demonstrate that the lipopeptide does notexchange out of lipid bilayers into potential “target” membranes, whichwould complicate interpretation of the lipid mixing results. Thetransfer of Lipo-AcE4K was investigated by incubating POPC LUVscontaining 10 mol % Lipo-AcE4K added after vesicle formation with alarge excess of POPC MLVs. The MLVs were separated from LUVs bycentrifugation. A comparison of the peptide contents of theLipo-AcE4K-bearing LUVs before and after incubation with MLVs is givenin FIG. 44A. It is clear that there is no exchange of Lipo-AcE4K out ofthe LUV population when incubated with MLVs. A small increase inmeasured peptide content after the incubation can be attributed tointerference in the assay caused by phospholipid as shown in the POPCcontrol.

[0343] Given this result, the ability of POPC vesicles containingLipo-AcE4K to fuse with POPC membranes lacking the lipopeptide wasinvestigated. As seen in FIG. 44B, very little lipid mixing is observedwhen only one liposome population contains 5 mol % Lipo-AcE4K comparedto the assay when the lipopeptide is present in both membranes. Therewas only a small difference in this result when the Lipo-AcE4K wasincorporated into the fluorescently labeled population rather theunlabeled population, reflecting the probability differences resultingfrom the mixing ratio of 1:3 labeled to unlabeled.

[0344] e. Fusion and Leakage in EPC/Chol Vesicles

[0345] In order to achieve higher levels of membrane fusion, we havelooked at the effect of Lipo-AcE4K on the stability of EPC/Chol (55:45)LUVs which more closely approximate the lipid composition of biologicalmembranes. EPC is a naturally-occurring mixture of phosphatidylcholinespecies bearing a variety of fatty acyl chains, and it consistspredominantly of POPC. While the addition of cholesterol to phospholipidbilayers decreases membrane permeability by effecting tighter packinglipids, cholesterol can also promote membrane fusion by inducing theformation of nonbilayer lipid phases.

[0346] As shown in FIG. 46, Lipo-AcE4K at a concentration of 5 or 10 mol% is more effective at promoting lipid mixing in EPC/Chol (55:45) LUVsthan in either EPC or POPC LUVs (see, FIG. 44A). Again, a transientrapid increase in fluorescence is observed; however, higher levels offluorescence are achieved, and lipid mixing continues at a reduced ratefor the duration of the assay. Interestingly, the corresponding ANTS-DPXleakage results for EPC/Chol liposomes, given in FIG. 46B, indicatelower levels of leakage at all concentrations of Lipo-AcE4K than wereobserved for POPC (FIG. 44B) or EPC (only 10 mol % Lipo-AcE4K datashown). It is remarkable that the inclusion of cholesterol appears toincrease the destabilization caused by the lipopeptide, perhaps by thepromotion of nonbilayer structures, while reducing the permeability ofthe destabilized membranes to the aqueous medium. However, leakage ofvesicle contents remains substantial, and in no case was contents mixingof vesicles observed.

[0347] f. Effects of Transbilayer Distribution of Lipo-AcE4K

[0348] Increased levels of lipid mixing were observed in EPC/Chol(55:45) vesicles when Lipo-AcE4K was present on both the inner and outermonolayers of the liposomes. This was achieved by adding the lipopeptideto the lipid preparation prior to freeze-drying, hydration, andextrusion. As shown in FIG. 9, lipid mixing between LUVs containing 10mol % Lipo-AcE4K prepared by this method gave values of ΔF/ΔF_(max)approaching 60% at 5 minutes. Furthermore, mixing these LUVs and anEPC/Chol (55:45) preparation lacking lipopeptide also resulted insubstantial membrane fusion. In contrast, LUVs prepared as before with10 mol % Lipo-AcE4K only on the outer monolayer could fuse withthemselves, but not with EPC/Chol LUVs.

[0349] While the Lipo-AcE4K on the interior of vesicles is unable topenetrate the target membrane, it can apparently play a role in furtherdestabilizing the membrane in which it is present. Presumably, this isas a result of reduction of the pH in the vesicle interior arising fromthe leakage induced by initial membrane destabilization.

[0350] g. Freeze-Fracture Electron Microscopy

[0351] The destabilization of lipid bilayers and lipid mixing induced byLipo-AcE4K can also be seen in freeze-fracture micrographs shown in FIG.48. EPC/Chol (55:45) samples without Lipo-AcE4K or those bearingLipo-AcE4K at pH 7.5 give smooth fracture surfaces and have sizedistributions typical of those normally observed for LUVs extrudedthrough 100 nm filters. Samples with 10 mol % Lipo-AcE4K at pH 5.0,however, have larger lipid structures, indicating fusion of liposomes.In addition, many of these larger structures exhibit rough surfaceswhich are believed to arise from penetration of the peptide portion ofLipo-AcE4K into the membrane. Furthermore, a large proportion of thesevesicles are cross-fractured, indicating that the pH-induced insertionof the lipopeptide does indeed disrupt bilayer structure and stability.The limited size increase observed is consistent with the transientdestabilization found in the lipid mixing and contents mixing assays.

[0352] h. Lipid Mixing with Erythrocyte Ghost Membranes

[0353] Ultimately, the destabilizing properties of Lipo-AcE4K directedtoward biological membranes are of greatest interest. As a model forsuch systems, lipid mixing between EPC/Chol (55:45) liposomes containingLipo-AcE4K with erythrocyte ghost membranes has been studied. The ghostpreparation used here included 1 mM MgSO₄ in the lysis and washingbuffers as described by Stock and Kant, supra (1974). While this resultsin the retention of a small amount of hemoglobin within the cells, itensures rapid resealing and membrane integrity. Analysis forglycerol-3-phosphate dehydrogenase activity as described by Sigmaconfirmed the formation of sealed, right-side-out ghosts which was notthe case for a preparation without 1 mM MgSO₄ (data not shown).

[0354] Fluorescence lipid mixing assays with EPC/Chol erythrocyte ghostswere performed using two different liposome preparations. First,Lipo-AcE4K was added to EPC/Chol (55:45) LUVs from DMSO stock solutionleading to incorporation of the lipopeptide into the outer monolayers ofthe LUVs at a concentration of approximately 10 mol % relative tosurface-exposed lipid. Addition of erythrocyte ghosts to thispreparation gave limited lipid mixing (ΔF/ΔF_(max)˜2%) when the pH wasdecreased to 5.0 (FIG. 49). This result is in agreement with that shownabove (FIG. 46(B)) in which incorporation of Lipo-AcE4K into the outermonolayer of one population of vesicles was insufficient to givesubstantial lipid mixing with a second population of membranes. A secondliposome preparation with 10 mol % Lipo-AcE4K in EPC/Chol (55:45),incorporating lipopeptide into both inner and outer monolayers, gavemuch higher levels of lipid mixing with erythrocyte ghosts at pH 5.0,ΔF/ΔF_(max) values approaching 40% at 5 minutes.

[0355] Finally, lipid mixing with erythrocyte ghosts was alsodemonstrated by fluorescence microscopy. The fluorescently labeledliposome preparations from the lipid-mixing assays were also used forthis procedure, since they contain Rh-PE at a concentration which isgreater than 80% self-quenching. The NBD-PE fluorescence is masked by ared transmission filter and is also readily photo-bleached under theconditions used here. This permits the detection of lipid mixing betweenlabeled liposomes and erythrocyte ghosts as an increase in Rh-PEfluorescence upon dilution into the target and reduced self-quenching.FIG. 50 illustrates the effect of decreasing the pH for a mixture ofLUVs prepared from EPC/Chol (55:45) co-lyophilized with 10 mol %Lipo-AcE4K and erythrocyte ghosts. Phase contrast micrographs are shownwith corresponding fluorescence images at pH 7.5 and 5.0. While theappearance of Rh-PE fluorescence in the erythrocyte membranes is themost striking effect of lowering the pH, aggregation of the erythrocyteghosts is also apparent. Interestingly, substantial levels of Rh-PEfluorescence and aggregation were also observed for EPC/Chol (55:45)LUVs pre-incubated with 10 mol % Lipo-AcE4K (i.e., outer monolayeronly), although a much smaller increase in NBD-PE fluorescence wasobserved in the quantitative assay. Labeled EPC/Chol (55:45) LUVswithout lipopeptide gave no lipid mixing or aggregation with erythrocyteghosts at pH 5.0 (not shown). These micrographs were obtained using verysmall sample volumes (˜5 μl) spread very thinly using large cover slipsin order to arrest the erythrocyte ghosts without using high lipidconcentrations or mounting solutions that can quench fluorescence. As aresult, the majority of the membranes appear flattened, and lysis of themembranes begins to occur after several minutes.

[0356] 3. EXPERIMENTAL FINDINGS

[0357] The free peptide only adopts an a-helical structure at low pH andonly in the presence of lipid vesicles. This behavior is very similar tothe wild-type (X31) and E4 peptides as studied by Rafalski, et al.,supra (1991), except that they observed a low level of helical structureeven at neutral pH. However, in that study, SUVs were used in the CDexperiments, where LUVs have been used here. The higher curvature ofsmall vesicles probably promotes hydrophobic interactions at pH 7.5 thatare not observed in essentially planar LUV membranes. It is clear fromthe CD and Tryptophan fluorescence results that AcE4K requiresneutralization of at least some of its acidic residues before it willinteract with a lipid bilayer.

[0358] Coupling the peptide AcE4K to a lipid anchor effects thestructure adopted by the peptide in the presence of lipid vesicles aswell as the extent of its interactions with the lipid bilayer as afunction of pH. The CD spectra of Lipo-AcE4K indicate that the anchoredpeptide adopts an a-helical structure at both neutral and acidic pH incontrast to the free peptide which is only a-helical at low pH in thepresence of liposomes. This suggests that constraints imposed by thelipid anchor induce some peptide structure at neutral pH but prevent anyfurther structural changes upon neutralization of acidic residues.However, the tryptophan fluorescence maxima indicate that the anchoredpeptide clearly experiences a more hydrophobic environment uponacidification of the medium compared to its uncoupled counterpart. Basedon the lipid-mixing and electron microscopy results presented here, webelieve that this hydrophobic environment results from increasedpenetration of Lipo-AcE4K into the lipid bilayer and not simply from aloss of charge on neighboring residues or the formation of lipopeptidecomplexes.

[0359] Lipo-ACE4K destabilizes POPC and EPC/Chol lipid vesicles atmildly acidic pH, but not at pH 7.5. The extent of destabilizationdepends not only on pH and peptide concentration, but also on membranecomposition and, at least in some preparations, on the transbilayerdistribution of peptide. Membrane destabilization was determinedquantitatively by lipid mixing and leakage of vesicle contents. Suchresults are very different from those reported for similar peptides.Duzgunes & Shavnin (1992) used a 17 amino acid peptide from theN-terminus of HA2X31 wild-type sequence (refer to wt sequence reportedabove) and found that it gave extensive leakage for EPC LUVs at bothneutral and low pH and no lipid mixing under any conditions. Rafalski,et al., supra, (1991) reported pH-dependent leakage for the 20 aminoacid wt and for E4, but no lipid mixing in either POPC or POPC/Cholvesicles. It is clear that features such as peptide length, acidity, andmembrane-anchoring all influence the membrane-destabilizing ability ofthese peptides.

[0360] The lipid mixing and leakage results presented here indicateextensive but short-lived membrane destabilization by 1 to 10 mol. %Lipo-AcE4K in both POPC and EPC/Chol vesicles. The rapid loss indestabilizing capacity may be due to the formulation of amembrane-stable conformation, a re-orientation of the lipopeptide, orthe formation of stabilizing oligomeric complexes upon interaction withthe lipid bilayer. The event appears to be accompanied by membranepenetration of the anchored peptide.

[0361] In addition to lipopeptide concentration, the extent of lipidmixing and leakage depend upon the lipid composition of the membranesinvolved. Lipo-AcE4K gave higher levels of lipid mixing in EPC/Chol(55:45) LUVs than in EPC or POPC LUVs. We tentatively attribute thisdifference to the ability of cholesterol to promote non-bilayerintermediates leading to membrane fusion [26]. Curiously, the increasesin lipid mixing were accompanied by decreases in the extent of leakageat all lipopeptide concentrations. While cholesterol is known to reducethe permeability of phospholipid membranes, it was not expected toexhibit this property while also promoting lipid mixing.

[0362] The transbilayer distribution of the lipopeptide also influencesthe degree of lipid mixing observed. When Lipo-AcE4K was added topreformed vesicles, lipid mixing was only substantial between vesiclepopulations that each contained the lipopeptide. Very little lipidmixing was observed when only one population contained as much as 10 mol% Lipo-AcE4K. This result suggests that when Lipo-AcE4K is incorporatedinto the outer membranes of lipid vesicles in this way, it can onlypenetrate and destabilize the membrane in which it is anchored.Alternatively, if it inserts into the membrane of target vesicles, itdoes not destabilize the target vesicles sufficiently to promote lipidmixing between the two populations. In caution it should be noted thatfusion with the labeled population of lipopeptide-bearing vesicles whichcannot be detected by the assay may simply be the predominant processand that lipid mixing with a second population would be promoted undermore constrained circumstances, e.g., with an endosome.

[0363] Incorporation of Lipo-AcE4K into both inner and outer leaflets ofEPC/Chol (55:45) LUVs not only gave higher levels of lipid mixingbetween populations of vesicles containing the lipopeptide , but alsocaused these vesicles to fuse with liposomes lacking Lipo-AcE4K and witherythrocyte ghosts at low pH. The increase in lipid mixing provided bythe presence of lipopeptide on the inner surface of the vesicle membranemust arise from the capability of Lipo-AcE4K to destabilize the membranein which it is anchored since in this case it is unable to insert intoan external lipid bilayer.

[0364] Fusion of EPC/Chol (55:45) LUVs containing 10 mol % Lipo-AcE4Kwas also observed by freeze-fracture electron microscopy. At pH 5.0,large lipid vesicles with diameters of several hundred nanometers wereformed. This limited size increase, compared to the extensive fusedstructures found in Ca²⁺-induced fusion of negatively charged liposomes,is consistent with the transient destabilization indicated by the lipidmixing and contents mixing results. The freeze-fracture micrographs alsoshow very rough lipid surfaces and extensive cross-fracturing, both ofwhich can be attributed to destabilization of the membrane structure bythe lipopeptide. Fluorescence microscopy of erythrocyte ghosts incubatedwith these liposomes indicated not only pH-dependent lipid mixing withbut also aggregation of the ghosts at low pH. No Rh-PE fluorescence wasobserved in the ghosts at pH 7.5 for either preparation or for EPC/CholLUVs without Lipo-AcE4K at either pH 7.5 or pH 5.0.

[0365] In summary, the lipopeptide Lipo-AcE4K forms stable bilayers inPOPC and EPC/Chol LUVs at concentrations up to 10 mol % at pH 7.5.Destabilization of these lipid vesicles can be induced by decreasing thepH below 6.0 which corresponds to the conditions under which the viralprotein, influenza HA, from which it is derived causes membrane fusion.This membrane destabilization not only results in extensive leakage ofliposomal contents, as has been demonstrated with a variety of otherviral fusion peptides and synthetic amphipathic helicies, but also inlipid mixing of LUVs as determined by fluorescent lipid probe dilution,and coalescence of lipid membranes shown by freeze-fracture electronmicroscopy. The extent of lipid mixing depends on pH, membranecomposition, and the concentration of the lipopeptide as well as on itsdistribution between the membrane leaflets. Addition of 10 mol %Lipo-AcE4K to EPC/Chol (55:45) LUVs- gave lipid mixing with erythrocyteghosts, the first example of fusion induced by a membrane-anchoredfusion peptide with a biological membrane.

[0366] It is to be understood that the above description is intended tobe illustrative and not restrictive. Many embodiments will be apparentto those of skill in the art upon reading the above description. Thescope of the invention should, therefore, be determined not withreference to the above description, but should instead be determinedwith reference to the appended claims, along with the full scope ofequivalents to which such claims are entitled. The disclosures of allarticles and references, including patent applications and publications,are incorporated herein by reference for all purpose.

What is claimed is:
 1. A lipopeptide, said lipopeptide comprising alipid covalently attached to a peptide by means of an amide bond.
 2. Thelipopeptide in accordance with claim 1 wherein said amide bond is formedbetween a carboxyl group of said lipid and an amino group of saidpeptide.
 3. The lipopeptide in accordance with claim 2 wherein saidamino group is the primary amino group of a lysine residue at theC-terminus of said peptide.
 4. The lipopeptide in accordance with claim1 wherein said lipid is a diacylglycerol.
 5. The lipopeptide inaccordance with claim 4 wherein said diacylglycerol is a member selectedfrom the group consisting of 1,2-distearoyl-sn-glycerol,1,2-dioleoyl-sn-glycerol, and 1,2-dipalmitoyl-sn-glycerol.
 6. Thelipopeptide in accordance with claim 1 wherein said peptide is a fusionpeptide.
 7. The lipopeptide in accordance with claim 6 wherein saidfusion peptide has a lysine residue at the C-terminus.
 8. Thelipopeptide in accordance with claim 6 wherein said fusion peptidecomprises the following amino acid sequence Ac-GLFEAIAGFIENGWEGMIDGK andconservative modifications thereof.
 9. The lipopeptide in accordancewith claim 6 wherein said fusion peptide is a member selected from thegroup consisting of Ac-GLFEAIAGFIENGWEGMIDGK;WEAALAEALAEALAEHLAEALAEALEALAA; GGYCLTRWMLIEAELKCFGNTAV;GGYCLTKWMILAAELKCFGNTAV; GGYCLEKWMIVASELKCFGNTAI;GGYCLEQWAIIWAGLKCFDNTVM; GLFEALAEFIEGGWEGLIEG; GLFEAIAEFLEAIABFLEG;GWEGLIEGIEGGWEGLIEG; GLFEALAEFIPGGWEGLIEG; GLLEELLELLEELWEELLEG;Ac-LARLLARLLARL-NHCH₃; Ac-LARLLPRLLARL-NHCH₃; Ac-LPRLLPRLLARL-NHCH₃;Ac-LPRLLPRLLPRL-NHCH₃; FEAALAEALAEALA; Myr-FEAALAEALAEALA;WEAAKAEAKAEAKAC; and poly(Glu-Aib-Leu-Aib)

wherein: Myr represents myristic acid; and Aib represents2-aminoisobutyric acid; and conservative modifications thereof.
 10. Alipopeptide in accordance with claim 1 wherein said lipid is1,2-distearoyl-sn-glycerol and said fusion peptide comprises thefollowing amino acid sequence Ac-GLFEAIAGFIENGWEGMIDGK and conservativemodifications thereof.
 11. A pharmaceutical composition for introducinga therapeutic compound into a cell of a host, comprising: a liposomecontaining a lipopeptide, said lipopeptide comprising a lipid covalentlyattached to a peptide by means of an amide bond; a therapeutic compoundcontained in said liposome; and a pharmaceutically acceptable carrier.12. The pharmaceutical composition in accordance with claim 11 whereinsaid therapeutic compound is contained in the aqueous interior of theliposome.
 13. The pharmaceutical composition in accordance with claim 11wherein said therapeutic compound is contained in the membrane of theliposome.
 14. The pharmaceutical composition in accordance with claim 11wherein said therapeutic compound is a nucleic acid molecule.
 15. Thepharmaceutical composition in accordance with claim 14, wherein saidnucleic acid molecule is DNA.
 16. The pharmaceutical composition inaccordance with claim 14 wherein said nucleic acid molecule is RNA. 17.The pharmaceutical composition in accordance with claim 11 wherein saidtherapeutic compound is a peptide or protein.
 18. The pharmaceuticalcomposition in accordance with claim 11 wherein said liposome furthercomprises cholesterol.
 19. A polymer having the general structure:[X—Y]_(n) in which: X is a trifunctional substrate, wherein at least oneof the functional groups is a carboxyl group or a protected carboxylgroup; Y is ethylene glycol; and n is an integer having a value rangingfrom 1 to
 20. 20. A polymer in accordance with claim 19 wherein saidethylene glycol is a member selected from the group consisting ofdi(ethylene glycol), tri(ethylene glycol), tetra(ethylene glycol),penta(ethylene glycol), hexa(ethylene glycol), hexa(ethylene glycol) andocta(ethylene glycol).
 21. A polymer in accordance with claim 19 whereinX is L-glutamic acid; and Y is tetra(ethylene glycol).
 22. A polymerhaving the general structure: [X—Y—Z]_(n) in which: X and Z areindependently selected and are trifunctional substrates wherein at leastone of the functional groups of each of the trifunctional substrates isa carboxyl group or a protected carboxyl group; Y is ethylene glycol;and n is an integer having a value ranging from 1 to
 20. 23. A polymerin accordance with claim 22 wherein said ethylene glycol is a memberselected from the group consisting of di(ethylene glycol), tri(ethyleneglycol), tetra(ethylene glycol), penta(ethylene glycol), hexa(ethyleneglycol), hexa(ethylene glycol) and octa(ethylene glycol).
 24. A polymerin accordance with claim 22 wherein X is L-glutamic acid; and Y istetra(ethylene glycol).
 25. A pharmaceutical composition for introducinga therapeutic compound into a cell of a host, comprising: a liposomecontaining a pH-sensitive fusogenic polymer, said pH-sensitive fusogenicpolymer selected from the group consisting of [X—Y]_(n) and [X—Y—Z]_(n)in which: X is a trifunctional substrate, wherein at least one of thefunctional groups is a carboxyl group or a protected carboxyl group; Zis a trifunctional substrate, wherein at least one of the functionalgroups is a carboxyl group or a protected carboxyl group Y is ethyleneglycol; and n is an integer having a value ranging from 1 to 20; atherapeutic compound contained in said liposome; and a pharmaceuticallyacceptable carrier.